DNA Diagnostics Based on Mass Spectrometry

ABSTRACT

Fast and highly accurate mass spectrometry-based processes for detecting a particular nucleic acid sequence in a biological sample are provided. Depending on the sequence to be detected, the processes can be used, for example, to diagnose a genetic disease or chromosomal abnormality; a predisposition to a disease or condition, infection by a pathogenic organism, or for determining identity or heredity.

RELATED APPLICATIONS

For U.S. National Stage purposes, this application is acontinuation-in-part of U.S. application Ser. No. 08/744,481, filed Nov.6, 1996, to Köster, entitled “DNA DIAGNOSTICS BASED ON MASSSPECTROMETRY”. This application is also a continuation-in-part of U.S.application Ser. Nos. 08/744,590, 08/746,036, 08/746,055, 08/786,988,08/787,639, 08/933,792 and U.S. application Serial No. atty dkt. no.7352-2001B, filed Oct. 8, 1997, which is a continuation-in-part of U.S.application Ser. Nos. 08/746,055, 08/786,988 and 08/787,639. Forinternational purposes, benefit of priority is claimed to each of theseapplications.

This application is related to U.S. patent application Ser. No.08/617,256 filed on Mar. 18, 1996, which is a continuation-in-part ofU.S. application Ser. No. 08/406,199, filed Mar. 17, 1995, now U.S. Pat.No. 5,605,798, and is also related U.S. Pat. Nos. 5,547,835 and5,622,824.

Where permitted the subject matter of each of the above-noted patentapplications and the patent is herein incorporated in its entirety.

BACKGROUND OF THE INVENTION Detection of Mutations

The genetic information of all living organisms (e.g., animals, plantsand microorganisms) is encoded in deoxyribonucleic acid (DNA). Inhumans, the complete genome is contains of about 100,000 genes locatedon 24 chromosomes (The Human Genome, T. Strachan, BIOS ScientificPublishers, 1992). Each gene codes for a specific protein, which afterits expression via transcription and translation, fulfills a specificbiochemical function within a living cell. Changes in a DNA sequence areknown as mutations and can result in proteins with altered or in somecases even lost biochemical activities; this in turn can cause geneticdisease. Mutations include nucleotide deletions, insertions oralterations (i.e. point mutations). Point mutations can be either“missense”, resulting in a change in the amino acid sequence of aprotein or “nonsense” coding for a stop codon and thereby leading to atruncated protein.

More than 3000 genetic diseases are currently known (Human GenomeMutations, D. N. Cooper and M. Krawczak, BIOS Publishers, 1993),including hemophilias, thalassemias, Duchenne Muscular Dystrophy (DMD),Huntington's Disease (HD), Alzheimer's Disease and Cystic Fibrosis (CF).In addition to mutated genes, which result in genetic disease, certainbirth defects are the result of chromosomal abnormalities such asTrisomy 21 (Down's Syndrome), Trisomy 13 (Patau Syndrome), Trisomy 18(Edward's Syndrome), Monosomy X (Turner's Syndrome) and other sexchromosome aneuploidies such as Klienfelter's Syndrome (XXY). Further,there is growing evidence that certain DNA sequences may predispose anindividual to any of a number of diseases such as diabetes,arteriosclerosis, obesity, various autoimmune diseases and cancer (e.g.,colorectal, breast, ovarian, lung).

Viruses, bacteria, fungi and other infectious organisms contain distinctnucleic acid sequences, which are different from the sequences containedin the host cell. Therefore, infectious organisms can also be detectedand identified based on their specific DNA sequences.

Since the sequence of about 16 nucleotides is specific on statisticalgrounds even for the size of the human genome, relatively short nucleicacid sequences can be used to detect normal and defective genes inhigher organisms and to detect infectious microorganisms (e.g.,bacteria, fungi, protists and yeast) and viruses. DNA sequences can evenserve as a fingerprint for detection of different individuals within thesame species (see, Thompson, J. S, and M. W. Thompson, eds., Genetics inMedicine, W.B. Saunders Co., Philadelphia, Pa. (1991)).

Several methods for detecting DNA are currently being used. For example,nucleic acid sequences can be identified by comparing the mobility of anamplified nucleic acid fragment with a known standard by gelelectrophoresis, or by hybridization with a probe, which iscomplementary to the sequence to be identified. Identification, however,can only be accomplished if the nucleic acid fragment is labeled with asensitive reporter function (e.g., radioactive (³²P, ³⁵S), fluorescentor chemiluminescent). Radioactive labels can be hazardous and thesignals they produce decay over time. Non-isotopic labels (e.g.,fluorescent) suffer from a lack of sensitivity and fading of the signalwhen high intensity lasers are being used. Additionally, performinglabeling, electrophoresis and subsequent detection are laborious,time-consuming and error-prone procedures. Electrophoresis isparticularly error-prone, since the size or the molecular weight of thenucleic acid cannot be directly correlated to the mobility in the gelmatrix. It is known that sequence specific effects, secondary structureand interactions with the gel matrix are causing artifacts.

Use of Mass Spectrometry for Detection and Identification of NucleicAcids

Mass spectrometry provides a means of “weighing” individual molecules byionizing the molecules in vacuo and making them “fly” by volatilization.Under the influence of combinations of electric and magnetic fields, theions follow trajectories depending on their individual mass (m) andcharge (z). In the range of molecules with low molecular weight, massspectrometry has long been part of the routine physical-organicrepertoire for analysis and characterization of organic molecules by thedetermination of the mass of the parent molecular ion. In addition, byarranging collisions of this parent molecular ion with other particles(e.g., argon atoms), the molecular ion is fragmented forming secondaryions by the so-called collision induced dissociation (CID). Thefragmentation pattern/pathway very often allows the derivation ofdetailed structural information. Many applications of mass spectrometricmethods are known in the art, particularly in biosciences (see, e.g.,Methods in Enzymol., Vol. 193: “Mass Spectrometry” (J. A. McCloskey,editor), 1990, Academic Press, New York).

Because of the apparent analytical advantages of mass spectrometry inproviding high detection sensitivity, accuracy of mass measurements,detailed structural information by CID in conjunction with an MS/MSconfiguration and speed, as well as on-line data transfer to a computer,there has been interest in the use of mass spectrometry for thestructural analysis of nucleic acids. Recent reviews summarizing thisfield include K. H. Schram, “Mass Spectrometry of Nucleic AcidComponents,” Biomedical Applications of Mass Spectrometry 34: 203-287(1990); and P. F. Crain, “Mass Spectrometric Techniques in Nucleic AcidResearch,” Mass Spectrometry Reviews 9: 505-554 (1990); see, also U.S.Pat. No. 5,547,835 and U.S. Pat. No. 5,622,824).

Nucleic acids, however, are very polar biopolymers that are verydifficult to volatilize. Consequently, mass spectrometric detection hasbeen limited to low molecular weight synthetic oligonucleotides forconfirming an already known oligonucleotide sequence by determining themass of the parent molecular ion, or alternatively, confirming a knownsequence through the generation of secondary ions (fragment ions) viaCID in an MS/MS configuration using, in particular, for the ionizationand volatilization, the method of fast atomic bombardment (FAB massspectrometry) or plasma desorption (PD mass spectrometry). As anexample, the application of FAB to the analysis of protected dimericblocks for chemical synthesis of oligodeoxynucleotides has beendescribed (Köster et al. (1987) Biomed. Environ. Mass Spectrometry 14:111-116).

Other ionization/desorption techniques include electrospray/ion-spray(ES) and matrix-assisted laser desorption/ionization (MALDI). ES massspectrometry has been introduced by Fenn et al. (J. Phys. Chem. 88:4451-59 (1984); PCT Application No. WO 90/14148) and currentapplications are summarized in review articles (see, e.g., Smith et al.(1990) Anal. Chem. 62: 882-89 and Ardrey (1992), “Electrospray MassSpectrometry”, Spectroscopy Europe 4: 10-18). The molecular weights of atetradecanucleotide (see, Covey et al. (1988) The “Determination ofProtein, Oligonucleotide and Peptide Molecular Weights by Ionspray MassSpectrometry,” Rapid Commun. in Mass Spectrometry 2:249-256), and of a21-mer (Methods in Enzymol., 193, “Mass Spectrometry” (McCloskey,editor), p. 425, 1990, Academic Press, New York) have been published. Asa mass analyzer, a quadrupole is most frequently used. Because of thepresence of multiple ion peaks that all could be used for the masscalculation, the determination of molecular weights in femtomole amountsof sample is very accurate.

MALDI mass spectrometry, in contrast, can be attractive when atime-of-flight (TOF) configuration (see, Hillenkamp et al. (1990) pp49-60 in “Matrix Assisted UV-Laser Desorption/Ionization: A New Approachto Mass Spectrometry of Large Biomolecules,” Biological MassSpectrometry, Burlingame and McCloskey, editors, Elsevier SciencePublishers, Amsterdam) is used as a mass analyzer. Since, in most cases,no multiple molecular ion peaks are produced with this technique, themass spectra, in principle, look simpler compared to ES massspectrometry.

Although DNA molecules up to a molecular weight of 410,000 daltons havebeen desorbed and volatilized (Williams et al., “Volatilization of HighMolecular Weight DNA by Pulsed Laser Ablation of Frozen AqueousSolutions,” Science 246: 1585-87 (1989)), this technique had only shownvery low resolution (oligothymidylic acids up to 18 nucleotides,Huth-Fehre et al. Rapid Commun. in Mass Spectrom., 6: 209-13 (1992); DNAfragments up to 500 nucleotides in length K. Tang et al., Rapid Commun.in Mass Spectrom., 8: 727-730 (1994); and a double-stranded DNA of 28base pairs (Williams et al., “Time-of-Flight Mass Spectrometry ofNucleic Acids by Laser Ablation and Ionization from a Frozen AqueousMatrix,” Rapid Commun. in Mass Spectrom., 4: 348-351 (1990)). JapanesePatent No. 59-131909 describes an instrument, which detects nucleic acidfragments separated either by electrophoresis, liquid chromatography orhigh speed gel filtration. Mass spectrometric detection is achieved byincorporating into the nucleic acids, atoms, such as S, Br, I or Ag, Au,Pt, Os, Hg, that normally do not occur in DNA.

Co-owned U.S. Pat. No. 5,622,824 describes methods for DNA sequencingbased on mass spectrometric detection. To achieve this, the DNA is bymeans of protection, specificity of enzymatic activity, orimmobilization, unilaterally degraded in a stepwise manner viaexonuclease digestion and the nucleotides or derivatives detected bymass spectrometry. Prior to the enzymatic degradation, sets of ordereddeletions that span a cloned DNA fragment can be created. In thismanner, mass-modified nucleotides can be incorporated using acombination of exonuclease and DNA/RNA polymerase. This permits eithermultiplex mass spectrometric detection, or modulation of the activity ofthe exonuclease so as to synchronize the degradative process. Co-ownedU.S. Pat. Nos. 5,605,798 and 5,547,835 provide methods for detecting aparticular nucleic acid sequence in a biological sample. Depending onthe sequence to be detected, the processes can be used, for example, inmethods of diagnosis. These methods, while broadly useful and applicableto numerous embodiments, represent the first disclosure of suchapplications and can be improved upon.

Therefore, it is an object herein to provided improved methods forsequencing and detecting DNA molecules in biological samples. It is alsoan object herein to provided improved methods for diagnosis of geneticdiseases, predispositions to certain diseases, cancers, and infections.

SUMMARY OF THE INVENTION

Methods of diagnosis by detecting and/or determining sequences ofnucleic acids that are based on mass spectrometry are provided herein.Methods are provided for detecting double-stranded DNA, detectingmutations and other diagnostic markers using MS analysis. In particular,methods for diagnosing neuroblastoma, detecting heredity relationships,HLA compatibility, genetic fingerprinting, detecting telomerase activityfor cancer diagnosis are provided.

In certain embodiments the DNA is immobilized on a solid support eitherdirectly or via a linker and/or bead. Three permutations of the methodsfor DNA detection in which immobilized DNA is used are exemplified.These include: (1) immobilization of a template; hybridization of theprimer; extension of the primer, or extension of the primer (singleddNTP) for sequencing or diagnostics or extension of the primer andEndonuclease degradation (sequencing); (2) immobilization of a primer;hybridization of a single stranded template; and extension of theprimer, or extension of the primer (single ddNTP) for sequencing ordiagnostics or extension of the primer and Endonuclease degradation(sequencing); (3) immobilization of the primer; hybridization of adouble stranded template; extension of the primer, or extension of theprimer (single ddNTP) for sequencing or diagnostics or extension of theprimer and Endonuclease degradation (sequencing).

In certain embodiments the DNA is immobilized on the support via aselectively cleavable linker. Selectively cleavable linkers include, butare not limited to photocleavable linkers, chemically cleavable linkersand an enzymatically (such as a restriction site (nucleic acid linker),a protease site) cleavable linkers. Inclusion of a selectively cleavablelinker expands the capabilities of the MALDI-TOF MS analysis because itallows for all of the permutations of immobilization of DNA forMALDI-TOF MS, the DNA linkage to the support through the 3′- or 5′-endof a nucleic acid; allows the amplified DNA or the target primer to beextended by DNA synthesis; and further allows for the mass of theextended product (or degraded product via exonuclease degradation) to beof a size that is appropriate for MALDI-TOF MS analysis (i.e., theisolated or synthesized DNA can be large and a small primer or a largeprimer sequence can be used and a small restriction fragment of a geneor single strand thereof hybridized thereto).

In a preferred embodiment, the selectively cleavable linker is achemical or photocleavable linker that is cleaved during the ionizingstep of mass spectrometry. Exemplary linkers include linkers containing,a disulfide group, a leuvinyl group, an acid-labile trityl group and ahydrophobic trityl group. In other embodiments, the enzymaticallycleavable linker can be a nucleic acid that is an RNA nucleotide or thatencodes a restriction endonuclease site. Other enzymatically cleavablelinkers include linkers that contain a pyrophosphate group, anarginine-arginine group and a lysine-lysine group. Other linkers areexemplified herein.

Methods for sequencing long fragments of DNA are provided. To performsuch sequencing, specific base terminated fragments are generated from atarget nucleic acid. The analysis of fragments rather than the fulllength nucleic acid shifts the mass of the ions to be determined into alower mass range, which is generally more amenable to mass spectrometricdetection. For example, the shift to smaller masses increases massresolution, mass accuracy and, in particular, the sensitivity fordetection. Hybridization events and the actual molecular weights of thefragments as determined by mass spectrometry provide sequenceinformation (e.g., the presence and/or identity of a mutation). In apreferred embodiment, the fragments are captured on a solid supportprior to hybridization and/or mass spectrometry detection. In anotherpreferred embodiment, the fragments generated are ordered to provide thesequence of the larger nucleic acid.

One preferred method for generating base specifically terminatedfragments from a nucleic acid is effected by contacting an appropriateamount of a target nucleic acid with an appropriate amount of a specificendonuclease, thereby resulting in partial or complete digestion of thetarget nucleic acid. Endonucleases will typically degrade a sequenceinto pieces of no more than about 50-70 nucleotides, even if thereaction is not run to full completion. In a preferred embodiment, thenucleic acid is a ribonucleic acid and the endonuclease is aribonuclease (RNase) selected from among: the G-specific RNase T₁, theA-specific RNase U₂, the A/U specific RNase PhyM, U/C specific RNase A,C specific chicken liver RNase (RNase CL3) or crisavitin. In anotherpreferred embodiment, the endonuclease is a restriction enzyme thatcleaves at least one site contained within the target nucleic acid.Another preferred method for generating base specifically terminatedfragments includes performing a combined amplification and base-specifictermination reaction (e.g., using an appropriate amount of a first DNApolymerase, which has a relatively low affinity towards thechain-terminating nucleotides resulting in an exponential amplificationof the target; and a polymerase with a relatively high affinity for thechain terminating nucleotide resulting in base-specific termination ofthe polymerization. Inclusion of a tag at the 5′ and/or 3′ end of atarget nucleic acid can facilitates the ordering of fragments.

Methods for determining the sequence of an unknown nucleic acid in whichthe 5′ and/or 3′ end of the target nucleic acid can include a tag areprovided. Inclusion of a non-natural tag on the 3′ end is also usefulfor ruling out or compensating for the influence of 3′ heterogeneity,premature termination and nonspecific elongation. In a preferredembodiment, the tag is an affinity tag (e.g., biotin or a nucleic acidthat hybridizes to a capture nucleic acid). Most preferably the affinitytag facilitates binding of the nucleic acid to a solid support. Inanother preferred embodiment, the tag is a mass marker (i.e. a marker ofa mass that does not correspond to the mass of any of the fournucleotides). In a further embodiment, the tag is a natural tag, such asa polyA tail or the natural 3′ heterogeneity that can result, forexample, from a transcription reaction.

Methods of sequence analysis in which nucleic acids have been replicatedfrom a nucleic acid molecule obtained from a biological sample arespecifically digested using one or more nucleases (deoxyribonucleasesfor DNA, and ribonucleases for RNA) are provided. The fragments capturedon a solid support carrying the corresponding complementary sequences.Hybridization events and the actual molecular weights of the capturedtarget sequences provide information on mutations in the gene. The arraycan be analyzed spot-by-spot using mass spectrometry. Further, thefragments generated can be ordered to provide the sequence of the largertarget fragment.

In another embodiment, at least one primer with a 3′-terminal base ishybridized to the target nucleic acid near a site where possiblemutations are to be detected. An appropriate polymerase and a set ofthree nucleoside triphosphates (NTPs) and the fourth added as aterminator are reacted. The extension reaction products are measured bymass spectrometry and are indicative of the presence and the nature of amutation. The set of three NTPs and one dd-NTP (or three NTPs and one3′-deoxy NTP), will be varied to be able to discriminate between severalmutations (including compound heterozygotes) in the target nucleic acidsequence.

Methods for detecting and diagnosing neoplasia/malignancies in a tissueor cell sample are provided. The methods rely on a telomeric repeatamplification protocol (TRAP)-MS assay and include the steps of:

-   -   a) obtaining a tissue or a cell sample, such as a clinical        isolate or culture of suspected cells;    -   b) isolating/extracting/purifying telomerase from the sample;    -   c) adding the telomerase extract to a composition containing a        synthetic DNA primer, which is optionally immobilized,        complementary to the telomeric repeat, and all four dNTPs under        conditions that result in telomerase specific extension of the        synthetic DNA;    -   d) amplifying the telomerase extended DNA products, preferably        using a primer that contains a “linker moiety”, such as a moiety        based on thiol chemistry or streptavidin;    -   e) isolating linker-amplified primers, such as by using a        complementary binding partner immobilized on a solid support;    -   f) optionally conditioning the DNA for crystal formation; and    -   g) performing MS by ionizing/volatizing the sample to detect the        DNA product.        Telomerase-specific extension is indicative of        neoplasia/malignancy. This method can be used to detect ect        specific malignancies. The use of MS to detect the DNA product        permits identification the extended product, which is indicative        of telomerase activity in the sample. If desired, the synthetic        DNA can be in the form an array.

Methods for detecting mutations are provided and the use thereofoncogenes and to thereby screen for transformed cells, which areindicative of neoplasia. Detection of mutations present in oncogenes areindicative of transformation. This method includes the steps of:

-   -   a) obtaining a biological sample;    -   b) amplifying a portion of the selected proto-oncogene that        includes a codon indicative of transformation, where one primer        has a linker moiety for immobilization;    -   c) immobilizing DNA via the linker moiety to a solid support,        optionally in the form of an array;    -   d) hybridizing a primer complementary to the proto oncogene        sequence that is upstream from the codon    -   e) adding 3dNTPs/1 ddNTP and DNA polymerase and extending the        hybridized primer to the next ddNTP location;    -   f) ionizing/volatizing the sample; and    -   g) detecting the mass of the extended DNA, whereby mass        indicates the presence of wild-type or mutant alleles. The        presence of a mutant allele at the codon is diagnostic for        neoplasia.        In an exemplary embodiment, extension-MS analysis is used detect        the presence of a mutated codon 634 in the retrovirus        (RET)-proto oncogene.

In another embodiment, methods for diagnosing diseases using reversetranscription and amplification of a gene expressed in transformedcells. In particular, a method for diagnosis of neuroblastoma usingreverse transcriptase (RT)-MS of tyrosine hydroxylase, which is acatecholamine biosynthetic enzyme that expressed in tumor cells, but notin tumor cells but not normal cells, such as normal bone marrow cells isprovided. The method includes the steps of:

-   -   a) obtaining a tissue sample;    -   b) isolating polyA RNA from the sample;    -   c) preparing a cDNA library using reverse transcription;    -   d) amplifying a cDNA product, or portion thereof, of the        selected gene, where one oligo primer has a linker moiety;    -   e) isolating the amplified product by immobilizing the DNA to        solid support via the linker moiety;    -   f) optionally conditioning the DNA:    -   g) ionizing/volatizing sample and detecting the presence of a        DNA peak that is indicative of expression of the selected gene.        For example, expression of the tyrosine hydroxylase gene is        indicative of neuroblastoma.

Also provided are methods of directly detecting a double-strandednucleic acid using MALDI-TOF MS. These methods include the steps of:

-   -   a) isolating a double stranded DNA of an appropriate size for MS        via amplification methods or formed by hybridization of        single-stranded DNA fragment;    -   b) preparing the double-stranded DNA for analysis under        conditions that increase the ratio of dsDNA:ssDNA in which the        conditions include one or all of the following: preparing        samples for analysis at reduced temperatures (i.e. 4° C.), and        using of higher DNA concentrations in the matrix to drive duplex        formation    -   c) ionizing/volatizing the sample of step b), where this step        uses low acceleration voltage of the ions to assist in        maintaining duplex DNA by, for example, adjusting laser power to        just above threshold irradiation for ionization, and    -   d) detecting the presence of the dsDNA of the appropriate mass.        In preferred embodiments, the matrix includes 3-hydroxypicolinic        acid. The detected DNA can be indicative of a genetic disorder,        genetic disease, genetic predisposition to a disease chromosomal        abnormalities. In other embodiments, the mass of the double        stranded DNA is indicative of the deletion, insertion, mutation.

A method designated primer oligo base extension (PROBE) is provided.This method uses a single detection primer followed by anoligonucleotide extension step to give products, which can be readilyresolved by MALDI-TOF mass spectrometry. The products differ in lengthby a number of bases specific for a number of repeat units or for secondsite mutations within the repeated region. The method is exemplifiedusing as a model system the AluVpA polymorphism in intron 5 of theinterferon-α receptor gene located on human chromosome 21, and the polyT tract of the splice acceptor site of intron 8 from the CFTR genelocated on human chromosome 7. The method is advantageously used forexample, for determining identity, identifying mutations, familialrelationship, HLA compatibility and other such markers using PROBE-MSanalysis of microsatellite DNA. In a preferred embodiment, the methodincludes the steps of:

-   -   a) obtaining a biological sample from two individuals;    -   b) amplifying a region of DNA from each individual that contains        two or more microsatellite DNA repeat sequences    -   c) ionizing/volatizing the amplified DNA;    -   d) detecting the presence of the amplified DNA and comparing the        molecular weight of the amplified DNA. Different sizes are        indicative of non-identity (i.e. wild-type versus mutation),        non-heredity or non-compatibility; similar size fragments        indicate the possibility identity, of familial relationship, or        HLA compatibility.

More than one marker may be examined simultaneously, primers withdifferent linker moieties are used for immobilization.

Another method loop-primer oligo base extension, designated LOOP-PROBE,for detection of mutations especially predominant disease causingmutations or common polymorphisms is provided. In a particularembodiment, this method for detecting target nucleic acid in a sample,includes the steps of:

-   -   a) amplifying a target nucleic acid sequence, such as β-globin,        in a sample, using (i) a first primer whose 5′-end shares        identity to a portion of the target DNA immediately downstream        from the targeted codon followed by a sequence that introduces a        unique restriction endonuclease site, such as CfoI in the case        of β-globin, into the amplicon and whose 3′-end primer is        self-complementary; and (ii) a second downstream primer that        contains a tag, such as biotin, for immobilizing the DNA to a        solid support, such as streptavidin beads;    -   c) immobilizing the double-stranded amplified DNA to a solid        support via the linker moiety;    -   d) denaturing the immobilized DNA and isolating the        non-immobilized DNA strand;    -   e) annealing the intracomplementary sequences in the 3′-end of        the isolated non-immobilized DNA strand, such that the 3′-end is        extendable by a polymerase, which annealing can be performed,        for example, by heating then and cooling to about 37° C., or        other suitable method;    -   f) extending the annealed DNA by adding DNA polymerase, 3        dNTPs/1 ddNTP, whereby the 3′-end of the DNA strand is extended        by the DNA polymerase to the position of the next ddNTP location        (i.e., to the mutation location);    -   g) cleaving the extended double stranded stem loop DNA with the        unique restriction endonuclease and removing the cleaved stem        loop DNA    -   i) (optionally adding a matrix) ionizing/volatizing the extended        product; and    -   j) detecting the presence of the extended target nucleic acid,        whereby the presence of a DNA fragment of a mass different from        wild-type is indicative of a mutation at the target codon(s).        This method eliminates one specific reagent for mutation        detection compared other methods of MS mutational analyses,        thereby simplifying the process and rendering it amenable to        automation. Also, the specific extended product that is analyzed        is cleaved from the primer and is therefore shorter compared to        the other methods. In addition, the annealing efficiency is        higher compared to annealing of an added primer and should        therefore generate more product. The process is compatible with        multiplexing and various detection schemes (e.g., single base        extension, oligo base extension and sequencing). For example,        the extension of the loop-primer can be used for generation of        short diagnostic sequencing ladders within highly polymorphic        regions to perform, for example, HLA typing or resistance as        well as species typing.

In another embodiment, a methods of detecting a target nucleic acid in abiological sample using RNA amplification is provided. In the method,the target is amplified the target nucleic acid, using a primer thatshares a region complementary to the target sequence and upstreamencodes a promoter, such as the T7 promoter. A DNA-dependent RNApolymerase and appropriate ribonucleotides are added to synthesize RNA,which is analyzed by MS.

Improved methods of sequencing DNA using MS are provided. In thesemethods thermocycling for amplification is used prior to MS analysis,thereby increasing the signal.

Also provide are primers for use in MS analyses. In particular, primers,comprising all or, for longer oligonucleotides, at least about 20,preferably about 16, bases of any of the sequence of nucleotidessequences set forth in SEQ ID NOs: 1-22, 24, 27-38, 41-86, 89, 92, 95,98, 101-110, 112-123, 126, 128, 129, and primers set forth in SEQ IDNOs: 280-287. The primers are unlabeled, and optionally include a massmodifying moiety, which is preferably attached to the 5′ end.

Other features and advantages of the methods provided herein will befurther described with reference to the following Figures, DetailedDescription and Claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a diagram showing a process for performing mass spectrometricanalysis on one target detection site (TDS) contained within a targetnucleic acid molecule (T), which has been obtained from a biologicalsample. A specific capture sequence (C) is attached to a solid support(SS) via a spacer (S). The capture sequence is chosen to specificallyhybridize with a complementary sequence on the target nucleic acidmolecule (T), known as the target capture site (TCS). The spacer (S)facilitates unhindered hybridization. A detector nucleic acid sequence(D), which is complementary to the TDS is then contacted with the TDS.Hybridization between D and the TDS can be detected by massspectrometry.

FIG. 1B is a diagram showing a process for performing mass spectrometricanalysis on at least one target detection site (here TDS 1 and TDS 2)via direct linkage to a solid support. The target sequence (T)containing the target detection site (TDS 1 and TDS 2) is immobilized toa solid support via the formation of a reversible or irreversible bondformed between an appropriate functionality (L′) on the target nucleicacid molecule (T) and an appropriate functionality (L) on the solidsupport. Detector nucleic acid sequences (here D1 and D2), which arecomplementary to a target detection site (TDS 1 or TDS 2) are thencontacted with the TDS. Hybridization between TDS 1 and D1 and/or TDS 2and D2 can be detected and distinguished based on molecular weightdifferences.

FIG. 1C is a diagram showing a process for detecting a wildtype (D^(wt))and/or a mutant (D^(mut)) sequence in a target (T) nucleic acidmolecule. As in FIG. 1A, a specific capture sequence (C) is attached toa solid support (SS) via a spacer (S). In addition, the capture sequenceis chosen to specifically interact with a complementary sequence on thetarget sequence (T), the target capture site (TCS) to be detectedthrough hybridization. If the target detection site (TDS) includes amutation, X, detection sites can be distinguished from wildtype by massspectrometry. Preferably, the detector nucleic acid molecule (D) isdesigned so that the mutation is in the middle of the molecule andtherefore would not lead to a stable hybrid if the wildtype detectoroligonucleotide (D^(wt)) is contacted with the target detector sequence,e.g., as a control. The mutation can also be detected if the mutateddetector oligonucleotide (D^(mut)) with the matching base at the mutatedposition is used for hybridization. If a nucleic acid molecule obtainedfrom a biological sample is heterozygous for the particular sequence(i.e. contain D^(wt) and D^(mut)), D^(wt) and D^(mut) will be bound tothe app and D^(mut) to be detected simultaneously.

FIG. 2 is a diagram showing a process in which several mutations aresimultaneously detected on one target sequence molecular weightdifferences between the detector oligonucleotides D1, D2 and D3 must belarge enough so that simultaneous detection (multiplexing) is possible.This can be achieved either by the sequence itself (composition orlength) or by the introduction of mass-modifying functionalities M1-M3into the detector oligonucleotide.

FIG. 3 is a diagram showing still another multiplex detection format. Inthis embodiment, differentiation is accomplished by employing differentspecific capture sequences which are position-specifically immobilizedon a flat surface (e.g., a ‘chip array’). If different target sequencesT1-Tn are present, their target capture sites TCS1-TCSn will interactwith complementary immobilized capture sequences C1-Cn. Detection isachieved by employing appropriately mass differentiated detectoroligonucleotides D1-Dn, which are mass differentiated either by theirsequences or by mass modifying functionalities M1-Mn.

FIG. 4 is a diagram showing a format wherein a predesigned targetcapture site (TCS) is incorporated into the target sequence usingnucleic acid (i.e., PCR) amplification. Only one strand is captured, theother is removed (e.g., based on the interaction between biotin andstreptavidin coated magnetic beads). If the biotin is attached to primer1 the other strand can be appropriately marked by a TCS. Detection is asdescribed above through the interaction of a specific detectoroligonucleotide D with the corresponding target detection site TDS viamass spectrometry.

FIG. 5 is a diagram showing how amplification (here ligase chainreaction (LCR)) products can be prepared and detected by massspectrometry. Mass differentiation can be achieved by the mass modifyingfunctionalities (M1 and M2) attached to primers (P1 and P4respectively). Detection by mass spectrometry can be accomplisheddirectly (i.e. without employing immobilization and target capturingsites (TCS)). Multiple LCR reactions can be performed in parallel byproviding an ordered array of capturing sequences (C). This formatallows separation of the ligation products and spot by spotidentification via mass spectrometry or multiplexing if massdifferentiation is sufficient.

FIG. 6A is a diagram showing mass spectrometric analysis of a nucleicacid molecule, which has been amplified by a transcription amplificationprocedure. An RNA sequence is captured via its TCS sequence, so thatwildtype and mutated target detection sites can be detected as above byemploying appropriate detector oligonucleotides (D).

FIG. 6B is a diagram showing multiplexing to detect two different(mutated) sites on the same RNA in a simultaneous fashion usingmass-modified detector oligonucleotides M1-D1 and M2-D2.

FIG. 6C is a diagram of a different multiplexing procedure for detectionof specific mutations by employing mass modified dideoxynucleoside or3′-deoxynucleoside triphosphates and an RNA dependent DNA polymerase.Alternatively, DNA dependent RNA polymerase and ribonucleotidephosphates can be employed. This format allows for simultaneousdetection of all four base possibilities at the site of a mutation (X).

FIG. 7A is a diagram showing a process for performing mass spectrometricanalysis on one target detection site (TDS) contained within a targetnucleic acid molecule (T), which has been obtained from a biologicalsample. A specific capture sequence (C) is attached to a solid support(SS) via a spacer (S). The capture sequence is chosen to specificallyhybridize with a complementary sequence on T known as the target capturesite (TCS). A nucleic acid molecule that is complementary to a portionof the TDS is hybridized to the TDS 5′ of the site of a mutation (X)within the TDS. The addition of a complete set of dideoxynucleosides or3′-deoxynucleoside triphosphates (e.g., pppAdd, pppTdd, pppCdd andpppGdd) and a DNA dependent DNA or RNA polymerase allows for theaddition only of the one dideoxynucleoside or 3′-deoxynucleosidetriphosphate that is complementary to X.

FIG. 7B is a diagram showing a process for performing mass spectrometricanalysis to determine the presence of a mutation at a potential mutationsite (M) within a nucleic acid molecule. This format allows forsimultaneous analysis of alleles (A) and (B) of a double stranded targetnucleic acid molecule, so that a diagnosis of homozygous normal,homozygous mutant or heterozygous can be provided. Allele A and B areeach hybridized with complementary oligonucleotides ((C) and (D)respectively), that hybridize to A and B within a region that includesM. Each heteroduplex is then contacted with a single strand specificendonuclease, so that a mismatch at M, indicating the presence of amutation, results in the cleavage of (C) and/or (D), which can then bedetected by mass spectrometry.

FIG. 8 is a diagram showing how both strands of a target DNA can beprepared for detection using transcription vectors having two differentpromoters at opposite locations (e.g., the SP6 and T7 promoter). Thisformat is particularly useful for detecting heterozygous targetdetections sites (TDS). Employing the SP6 or the T7 RNA polymerase bothstrands could be transcribed separately or simultaneously. Thetranscribed RNA molecules can be specifically captured andsimultaneously detected using appropriately mass-differentiated detectoroligonucleotides. This can be accomplished either directly in solutionor by parallel processing of many target sequences on an ordered arrayof specifically immobilized capturing sequences.

FIG. 9 is a diagram showing how RNA prepared as described in FIGS. 6, 7and 8 can be specifically digested using one or more ribonucleases andthe fragments captured on a solid support carrying the correspondingcomplementary sequences. Hybridization events and the actual molecularweights of the captured target sequences provide information on whetherand where mutations in the gene are present. The array can be analyzedspot by spot using mass spectrometry. DNA can be similarly digestedusing a cocktail of nucleases including restriction endonucleases.Mutations can be detected by different molecular weights of specific,individual fragments compared to the molecular weights of the wildtypefragments.

FIG. 10A shows UV spectra resulting from the experiment described in thefollowing Example 1. Panel i) shows the absorbance of the 26-mer beforehybridization. Panel ii) shows the filtrate of the centrifugation afterhybridization. Panel iii) shows the results after the first wash with 50mM ammonium citrate. Panel iv) shows the results after the second washwith 50 mM ammonium citrate.

FIG. 10B shows a mass spectrum resulting from the experiment describedin the following Example 1 after three washing/centrifugation steps.

FIG. 10C shows a mass spectrum resulting from the experiment describedin the following Example 1 showing the successful desorption of thehybridized 26-mer off of beads in accordance with the format depictedschematically in FIG. 1B.

FIG. 11 shows a mass spectrum resulting from the experiment described inthe following Example 1 showing the giving proof of an experiment asschematically depicted in FIG. 1B successful desorption of thehybridized 40-mer. The efficiency of detection suggests that fragmentsmuch longer than 40-mers can also be desorbed.

FIG. 12 shows a mass spectrum resulting from the experiment described inthe following Example 2 showing the successful desorption anddifferentiation of an 18-mer and 19-mer by electrospray massspectrometry, the mixture (top), peaks resulting from 18-mer emphasized(middle) and peaks resulting from 19-mer emphasized (bottom)

FIG. 13 is a graphic representation of the process for detecting theCystic Fibrosis mutation ΔF508 as described in Example 3.

FIG. 14 is a mass spectrum of the DNA extension product of a ΔF508homozygous normal of Example 3.

FIG. 15 is a mass spectrum of the DNA extension product of a ΔF508heterozygous mutant of Example 3.

FIG. 16 is a mass spectrum of the DNA extension product of a ΔF508homozygous normal of Example 3.

FIG. 17 is a mass spectrum of the DNA extension product of a ΔF508homozygous mutant of Example 3.

FIG. 18 is a mass spectrum of the DNA extension product of a ΔF508heterozygous mutant of Example 3.

FIG. 19 is a graphic representation of various processes for performingapolipoprotein E genotyping of Example 4.

FIG. 20 shows the nucleic acid sequence of normal apolipoprotein E(encoded by the E3 allele, FIG. 20B, SEQ ID NO: 130), and other isotypesencoded by the E2 and E4 alleles (FIG. 20A).

FIG. 21A shows a composite restriction pattern for various genotypes ofapolipoprotein E using the CfoI restriction endonuclease.

FIG. 21B shows the restriction pattern obtained in a 3.5% MetPhorAgarose Gel for various genotypes of apolipoprotein E.

FIG. 21C shows the restriction pattern obtained in a 12% polyacrylamidegel for various genotypes of apolipoprotein E.

FIG. 22A is a chart showing the molecular weights of the 91, 83, 72, 48and 35 base pair fragments obtained by restriction enzyme cleavage ofthe E2, E3 and E4 alleles of apolipoprotein E.

FIG. 22B is the mass spectrum of the restriction product of a homozygousE4 apolipoprotein E genotype.

FIG. 23A is the mass spectrum of the restriction product of a homozygousE3 apolipoprotein E genotype.

FIG. 23B is the mass spectrum of the restriction product of a E3/E4apolipoprotein E genotype.

FIG. 24 is an autoradiograph of Example 5 of a 7.5% polyacrylamide gelin which 10% (5 μl) of each amplified sample was loaded: sample M:pBR322 Alul digested; sample 1: HBV positive in serological analysis;sample 2: also HBV positive; sample 3: without serological analysis butwith an increased level of transaminases, indicating liver disease;sample 4: HBV negative containing HCV; sample 5: HBV posit-) negativecontrol; (+) positive control). Staining was done with ethidium bromide.

FIG. 25A is a mass spectrum of sample 1, which is HBV positive. Thesignal at 20754 Da represents the HBV related amplification product (67nucleotides, calculated mass: 20735 Da). The mass signal at 10390 Darepresents the [M+2H]²⁺ molecule ion (calculated: 10378 Da).

FIG. 25B is a mass spectrum of sample 3, which is HBV negativecorresponding to nucleic acid (i.e., PCR), serological and dot blotbased assays. The amplified product is generated only in trace amounts.Nevertheless it is unambiguously detected at 20751 Da (calculated mass:20735 Da). The mass signal at 10397 Da represents the [M+2H]²⁺ moleculeion (calculated: 10376 Da).

FIG. 25C is a mass spectrum of sample 4, which is HBV negative, but HCVpositive. No HBV specific signals were observed.

FIG. 26 shows a part of the E. coli lacI gene with binding sites of thecomplementary oligonucleotides used in the ligase chain reaction (LCR)of Example 6. Here the wildtype sequence is displayed. The mutantcontains a point mutation at bp 191 which is also the site of ligation(bold). The mutation is a C to T transition (G to A, respectively). Thisleads to a T-G mismatch with oligo B (and A-C mismatch with oligo C,respectively). Oligo A, B, C, and D sequences are set forth in SEQ IDNO: 9, 10, 11 and 12 respectively.

FIG. 27 is a 7.15% polyacrylamide gel of Example 6 stained with ethidiumbromide. M: chain length standard (pUC19DNA, MspI digested). Lane 1: LCRwith wildtype template. Lane 2: LCR with mutant template. Lane 3:(control) LCR without template. The ligation product (50 bp) was onlygenerated in the positive reaction containing wildtype template.

FIG. 28 is an HPLC chromatogram of two pooled positive LCRs.

FIG. 29 shows an HPLC chromatogram the same conditions but mutanttemplate were used. The small signal of the ligation product is due toeither template-free ligation of the educts or to a ligation at a (G-T,A-C) mismatch. The ‘false positive’ signal is significantly lower thanthe signal of ligation product with wildtype template depicted in FIG.28. The analysis of ligation educts leads to ‘double-peaks’ because twoof the oligonucleotides are 5′-phosphorylated.

FIG. 30 In (b) the complex signal pattern obtained by MALDI-TOF-MSanalysis of Pfu DNA-ligase solution of Example 6 is depicted. In (a) aMALDI-TOF-spectrum of an unpurified LCR is shown. The mass signal 67569Da probably represents the Pfu DNA ligase.

FIG. 31 shows a MALDI-TOF spectrum of two pooled positive LCRs (a). Thesignal at 7523 Da represents unligated oligo A (calculated: 7521 Da)whereas the signal at 15449 Da represents the ligation product(calculated: 15450 Da). The signal at 3774 Da is the [M+2H]²⁺ signal ofoligo A. The signals in the mass range lower than 2000 Da are due to thematrix ions. The spectrum corresponds to lane 1 in FIG. 27 and thechromatogram in FIG. 28. In (b) a spectrum of two pooled negative LCRs(mutant template) is shown. The signal at 7517 Da represents oligo A(calculated: 7521 Da).

FIG. 32 shows a spectrum of two pooled control reactions (with salmonsperm DNA as template). The signals in the mass range around 2000 Da aredue to Tween20, only oligo A could be detected, as expected.

FIG. 33 shows a spectrum of two pooled positive LCRs (a). Thepurification was done with a combination of ultrafiltration andstreptavidin DynaBeads as described in the text. The signal at 15448 Darepresents the ligation product (calculated: 15450 Da). The signal at7527 represents oligo A (calculated: 7521 Da). The signals at 3761 Da isthe [M+2H]²⁺ signal of oligo A, whereas the signal at 5140 Da is the[M+3H]²⁺ signal of the ligation product. In (b) a spectrum of two poolednegative LCRs (without template) is shown. The signal at 7514 Darepresents oligo A (calculated: 7521 Da).

FIG. 34 is a schematic presentation of the oligo base extension of themutation detection primer as described in Example 7, using ddTTP (A) orddCTP (B) in the reaction mix, respectively. The theoretical masscalculation is given in parenthesis. The sequence shown is part of theexon 10 of the CFTR gene (SEQ ID NO: 132) that bears the most commoncystic fibrosis mutation ΔF508 and more rare mutations A1507 as well asIle506Ser. The short sequencing products were produced using eitherddTTP (FIG. 34A; SEQ ID NOs: 133-135) or ddCTP (FIG. 34B; SEQ ID NOs:136-139).

FIG. 35 is a MALDI-TOF-MS spectrum recorded directly from precipitatedoligo base extended primers for mutation detection. The spectrum in (A)and (B), respectively show the annealed primer (CF508) without furtherextension reaction. Panel C displays the MALDI-TOF spectrum of the wildtype by using pppTdd in the extension reaction and D a heterozygoticextension products carrying the 506S mutation when using pppCdd asterminator. Panels E and F show a heterozygote with ΔF508 mutation withpppTdd and pppCdd as terminators in the extension reaction. Panels G andH represent a homozygous ΔF508 mutation with either pppTdd or pppCdd asterminators. The template of diagnosis is pointed out below eachspectrum and the observed/expected molecular mass are written inparenthesis.

FIG. 36 shows the portion of the sequence of pRFc1 DNA, which was usedas template for nucleic acid amplification in Example 8 of unmodifiedand 7-deazapurine containing 99-mer (SEQ ID NO: 141) and 200-mer (SEQ IDNO: 140) nucleic acids as well as the sequences of the 19-mer forwardprimer (SEQ ID NO: 17) and the two 18-mer reverse primers (SEQ ID NO:18).

FIG. 37 shows the portion of the nucleotide sequence of M13 mp18 RFIDNA, which was used in Example 8 for nucleic acid amplification ofunmodified and 7-deazapurine containing 103-mer nucleic acids (SEQ IDNO: 245). Also shown are nucleotide sequences of the 17-mer primers (SEQID NOs: 19 and 20) used in the nucleic acid amplification reaction.

FIG. 38 shows the result of a polyacrylamide gel electrophoresis ofamplified products described in Example 8 purified and concentrated forMALDI-TOF MS analysis. M: chain length marker, lane 1: 7-deazapurinecontaining 99-mer amplified product, lane 2: unmodified 99-mer, lane 3:7-deazapurine containing 103-mer and lane 4: unmodified 103-meramplified product.

FIG. 39: an autoradiogram of polyacrylamide gel electrophoresis ofnucleic acid (i.e., PCR) reactions carried out with 5′-[³²P]-labeledprimers 1 and 4. Lanes 1 and 2: unmodified and 7-deazapurine modified103-mer amplified product (53321 and 23520 counts), lanes 3 and 4:unmodified and 7-deazapurine modified 200-mer (71123 and 39582 counts)and lanes 5 and 6: unmodified and 7-deazapurine modified 99-mer (173216and 94400 counts).

FIG. 40 a) MALDI-TOF mass spectrum of the unmodified 103-mer amplifiedproducts (sum of twelve single shot spectra). The mean value of themasses calculated for the two single strands (31768 u and 31759 u) is31763 u. Mass resolution: 18. b) MALDI-TOF mass spectrum of7-deazapurine containing 103-mer amplified product (sum of three singleshot spectra). The mean value of the masses calculated for the twosingle strands (31727 u and 31719 u) is 31723 u. Mass resolution: 67.

FIG. 41: a) MALDI-TOF mass spectrum of the unmodified 99-mer amplifiedproduct (sum of twenty single shot spectra). Values of the massescalculated for the two single strands: 30261 u and 30794 u. b) MALDI-TOFmass spectrum of 7-deazapurine containing 99-mer amplified product (sumof twelve single shot spectra). Values of the masses calculated for thetwo single strands: 30224 u and 30750 u.

FIG. 42: a) MALDI-TOF mass spectrum of the unmodified 200-mer amplifiedproduct (sum of 30 single shot spectra). The mean value of the massescalculated for the two single strands (61873 u and 61595 u) is 61734 u.Mass resolution: 28. b) MALDI-TOF mass spectrum of 7-deazapurinecontaining 200-mer amplified product (sum of 30 single shot spectra).The mean value of the masses calculated for the two single strands(61772 u and 61714 u) is 61643 u. Mass resolution: 39.

FIG. 43: a) MALDI-TOF mass spectrum of 7-deazapurine containing 100-meramplified product with ribomodified primers. The mean value of themasses calculated for the two single strands (30529 u and 31095 u) is30812 u. b) MALDI-TOF mass spectrum of the amplified product afterhydrolytic primer-cleavage. The mean value of the masses calculated forthe two single strands (25104 u and 25229 u) is 25167 u. The mean valueof the cleaved primers (5437 u and 5918 u) is 5677 u.

FIG. 44 A-D shows the MALDI-TOF mass spectrum of the four sequencingladders obtained from a 39-mer template (SEQ ID NO: 23), which wasimmobilized to streptavidin beads via a 3′ biotinylation. A 14-merprimer (SEQ ID NO: 24) was used in the sequencing according to Example9.

FIG. 45 shows a MALDI-TOF mass spectrum of a solid phase sequencing of a78-mer template (SEQ ID NO: 25), which was immobilized to streptavidinbeads via a 3′ biotinylation. A 18-mer primer (SEQ ID NO: 26) and ddGTPwere used in the sequencing.

FIG. 46 shows a scheme in which duplex DNA probes with single-strandedoverhang capture specific DNA templates and also serve as primers forsolid phase sequencing.

FIG. 47 A-D shows MALDI-TOF mass spectra obtained from a sequencingreaction using 5′ fluorescent labeled 23-mer (SEQ ID NO: 29) annealed toa 3′ biotinylated 18-mer (SEQ ID NO: 30), leaving a 5-base overhang,which captured a 15-mer template (SEQ ID NO: 31) as described in Example9.

FIG. 48 shows a stacking fluorogram of the same products obtained fromthe reaction described in FIG. 47, but run on a conventional DNAsequencer.

FIG. 49 shows a MALDI-TOF mass spectrum of the sequencing ladder usingcycle sequencing as described in Example 1 generated from a biologicalamplified product as template and a 12mer (5′-TGC ACC TGA CTC-3′ (SEQ IDNO: 34)) sequencing primer. The peaks resulting from depurinations andpeaks which are not related to the sequence are marked by an asterisk.MALDI-TOF MS measurements were taken on a reflectron TOF MS. A.)Sequencing ladder stopped with ddATP; B.) Sequencing ladder stopped withddCTP; C.) Sequencing ladder stopped with ddGTP; D.) Sequencing ladderstopped with ddTTP.

FIG. 50 shows a schematic representation of the sequencing laddergenerated in FIG. 49 with the corresponding calculated molecular massesup to 40 bases after the primer. For the calculation, the followingmasses were used: 3581.4 Da for the primer, 312.2 Da for 7-deaza-dATP,304.2 Da for dTTP, 289.2 Da for dCTP and 328.2 Da for 7-deaza-dGTP.

FIG. 51 shows the sequence of the amplified 209 bp amplified product(SEQ ID NO: 261) within the β-globin gene, which was used as a templatefor sequencing. The sequences of the appropriate amplification primerand the location of the 12mer sequencing primer is also shown. Thissequence represents a homozygote mutant at the position 4 bases afterthe primer. In a wildtype sequence this T would be replaced by an A.

FIG. 52 shows a sequence (SEQ ID NO: 262) which is part of the intron 5of the interferon-receptor gene that bears the AluVpA polymorphism asfurther described in Example 11. The scheme presents the primer oligobase extension (PROBE) using ddGTP, ddCTP, or both for termination,respectively. The polymorphism detection primer (IFN) is underlined, thetermination nucleotides are marked in bold letters. The theoretical massvalues from the alleles found in 28 unrelated individuals and a fivemember family are given in the table. Both second site mutations foundin most 13 units allele, but not all, are indicated.

FIG. 53 shows the MALDI-TOF-MS spectra recorded directly formprecipitated extended cyclePROBE reaction products. Family study usingAluVpA polymorphism in intron 5 of the interferon-α receptor gene(Example 11).

FIG. 54 shows the mass spectra from PROBE products using ddC astermination nucleotide in the reaction mix. The allele with themolecular mass of approximately 11650 da from the DNA of the mother andchild 2 is a hint to a second site mutation within one of the repeatunits.

FIG. 55 shows a schematic presentation of the PROBE method for detectionof different alleles in the polyT tract at the 3′-end of intron 8 of theCFTR gene with pppCdd as terminator (Example 11) (SEQ ID NO: 263). Thesequence of the T5, T7 and T9 alleles are set forth in SEQ ID NOs: 264-6respectively.

FIG. 56 shows the MALDI-TOF-MS spectra recorded directly from theprecipitated extended PROBE reaction products. Detection of all threecommon alleles of the polyT tract at the 3′ end of Intron 8 of the CFTRgene. (a) T5/T9 heterozygous, (b) T7/T9 heterozygous (Example 11).

FIG. 57 shows a mass spectrum of the digestion of a 252-mer ApoE geneamplified product (ε3/ε3 genotype) as described in Example 12 using a)CfoI alone and b) CfoI plus RsaI. Asterisks: depurination peaks.

FIG. 58 shows a mass spectrum of the ApoE gene amplified product (ε3/ε3genotype) digested by CfoI and purified by a) single and b) doubleethanol/glycogen and c) double isopropyl alcohol/glycogenprecipitations.

FIG. 59 shows a mass spectrum of the CfoI/RsaI digest products from a)ε2/ε3, b) ε3/ε3, c) ε3/ε4, and d) ε4/ε4 genotypes. Dashed lines aredrawn through diagnostic fragments.

FIG. 60 shows a scheme for rapid identification of unknown ApoEgenotypes following simultaneous digestion of a 252-mer apo E geneamplified product by the restriction enzymes CfoI and RsaI.

FIG. 61 shows the multiplex (codons 112 and 158) mass spectrum PROBEresults for a) ε2/ε3, b) ε3/ε3, c) ε3/ε4, and d) ε4/ε4 genotypes. E:extension products; P: unextended primer. Top: codon 112 (SEQ ID NO:310) and 158 (SEQ ID NO: 311) regions, with polymorphic sites bold andprimer sequences underlined.

FIG. 62 shows a mass spectrum of a TRAP assay to detect telomeraseactivity (Example 13). The spectrum shows two of the primer signals ofthe amplified product TS primer at 5,497.3 Da (calc. 5523 Da) and thebiotinylated bioCX primer at 7,537.6 Da (calc. 7,537 Da) and the firsttelomerase-specific assay product containing three telomeric repeats at12,775.8 Da (calc. 12,452 Da) its mass is larger by one dA nucleotide(12,765 Da) due to extendase activity of Taq DNA polymerase.

FIG. 63 depicts the higher mass range of FIG. 62, i.e. the peak at12,775.6 Da represents the products with these telomeric repeats. Thepeaks at 20,322.1 Da is the result of a telomerase activity to formseven telomeric repeats (calc. 20,395 Da including the extension by onedA nucleotide). The peaks marked 1, 2, 3 and 4 contain a four telomericrepeats at 14,674 Da as well as secondary ion product.

FIG. 64 displays a MALDI-TOF spectrum of the RT-amplified product of thehuman tyrosine hydroxylase mRNA indicating the presence of neuroblastomacells (Example 14). The signal at 18,763.8 Da represents thenon-biotinylated single-stranded 61 mer of the nested amplified product(calc. 18,758.2 Da).

FIG. 65 (a) shows a schematic representation of a PROBE reaction for theRET proto-oncogene with a mixture of dATP, dCTP, dGTP, and ddTTP(Example 15). B represents biotin, through which the sense templatestrand is bound through streptavidin to a solid support. FIG. 65( b)shows the expected PROBE (SEQ ID NO: 53) products for ddT and ddAreactions for wildtype, C→T, and C→A antisense strands.

FIG. 66 shows the PROBE product mass spectra for (a) negative control,(b) Patient 1 being heterozygote (Wt/C→T) and (c) Patient 2 beingheterozygote (Wt/C→A), reporting average M_(r) values.

FIG. 67 shows the MALDI-FTMS spectra for synthetic analogs representingribo-cleaved RET proto-oncogene amplified products from (a) wildtype,(b) G→A, and (c) G→T homozygotes, and (d) wildtype/G→A, (e)wildtype/G→T, and (f) G→A/G→T heterozygotes, reporting masses of mostabundant isotope peaks.

FIG. 68 is a schematic representation of nucleic acid immobilization viacovalent bifunctional trityl linkers.

FIG. 69 is a schematic representation of nucleic acid immobilization viahydrophobic trityl linkers.

FIG. 70 shows a MALDI-TOF mass spectrum of a supernatant of the matrixtreated Dynabeads containing bound oligo (5′-iminobiotin-TGCACCTGACTC,SEQ ID NO: 56). An internal standard (CTGTGGTCGTGC, SEQ ID NO: 57) wasincluded in the matrix.

FIG. 71 shows a MALDI-TOF mass spectrum of a supernatant of the matrixtreated Dynabeads containing bound oligo (5′-iminobiotin-TGCACCTGACTC,SEQ ID NO: 56). An internal standard (CTGTGGTCGTGC, SEQ ID NO: 57) wasincluded in the matrix.

FIGS. 72A and 72B schematically depicts the steps involved with theLoop-primer oligo base extension (Loop-probe) reaction (SEQ ID NOs: 269and 273-275).

FIG. 73A shows a MALDI-TOF mass spectrum of a supernatant after CfoIdigest of a stem loop (SEQ ID NO: 276). FIG. 73B-D show MALDI-TOF massspectrum of different genotypes: HbA the wildtype genotype (74B (SEQ IDNO: 269)), HbC, a mutation of codon 6 of the β-globin gene which causessickle cell disease (74C), and HbS, a different mutation of codon 6 ofthe β-globin gene which causes sickle cell disease (74D).

FIG. 74 shows the nucleic acid sequence (SEQ ID NO: 277) of theamplified region of CKR-5. The underlined sequence corresponds to theregion homologous to the amplification primers. The dotted regioncorresponds to the 32 bp deletion.

FIG. 75 shows the sense primer ckrT7f (SEQ ID NO: 60). Being designed tofacilitate binding of T7-RNA polymerase and amplification of the CKR-5region to be analyzed, it starts with a randomly chosen sequence of 24bases, the T7 promoter sequence of 18 bases and the sequence homologousto CKR-5 of 19 bases.

FIG. 76 is a MALDI-TOF mass spectrum of the CKR-5 amplification product,which was generated as described in the following Example 21.

FIG. 77 is a positive ion UV-MALDI mass spectra of a synthetic RNA25-mer (5′-UCCGGUCUGAUGAGUCCGUGAGGAC-3′ SEQ ID NO: 62) digested withselected RNAses. For each enzyme 0.6 μl aliquots of the 4.5 μl assaycontaining a total of ca. 20 pmol of the RNA were fixed with 1.5 μlmatrix (3-HPA) for analysis. Fragments with retained 5′-terminus aremarked by different arrows, specific for the different RNAses, (Hahneret al., Proceedings of the 44^(th) ASMS Conference on Mass Spectrometryand Allied Topics, p. 983 (1996)).

FIG. 78 is an investigation of the specificity of the RNAses CL₃ andCusativin by positive ion UV-MALDI mass spectra of a synthetic RNA20-mer. Expected and/or observed cleavage sites are indicated by arrows.A, B, C indicate correct cleavage sites and corresponding singly cleavedfragments. Missing cleavages are designated by a question mark (?),unspecific cleavages by an X.

FIG. 79 shows the separation of a mixture of DNA molecules (12-mer,5′-biot. 19-mer, 22-mer and 5′-biot. 27-mer) with streptavidin-coatedmagnetic beads. a) positive ion UV-MALDI mass spectrum of 0.6 μl of amixture containing ca. 2-4 pmol of each species mixed with 1.5 μl matrix(3-HPA). b) same as a) but incubation of the mixture with magnetic beadsand subsequent release of the captured fragments.

FIG. 80 Elution of immobilized 5′ biotinylated 49 nt in vitro transcriptfrom the streptavidin-coated magnetic beads. Positive UV-MALDI massspectrum of the transcript prior to incubation with the magnetic beads(a). Spectra of the immobilized RNA transcript after elution with 95%formamide alone (b) and with various additives such as 10 mM EDTA (c),10 mM CDTA (d) and 25% ammonium hydroxide (e); EDTA and CDTA wereadjusted with 25% ammonium hydroxide to a pH of 8.

FIG. 81 Positive UV-MALDI mass spectra of the 5′ biotinylated 49 nt invitro transcript after RNAse U₂ digest for 15 minutes. a) Spectrum ofthe 25 ul assay containing ca. 100 pmol of the target RNA beforeseparation; b) spectrum after isolation of the 5′-biotinylated fragmentswith magnetic beads. Captured fragments were released by a solution of95% formamide containing 10 mM CDTA. 1 ul aliquots of the samples weremixed with 1.5 ul matrix (3-HPA) in both cases.

FIG. 82 schematically depicts detection of putative mutations in thehuman β-globin gene at codon 5 and 6 and at codon 30, and the IVS-1donor site, respectively, done in parallel. FIG. 82A (SEQ ID NOs: 68,281, 69, 283 and 287) shows amplification of genomic DNA using theprimers β2 and β11. The location of the primers and identification tagsas well as an indication of the wild type and mutant sequences areshown. FIG. 82B (SEQ ID NOs: 70, 288 and 289) shows analysis of bothsites in a simple Primer Reaction Oligo Base Extension (PROBE) usingprimers β-TAG1 (which binds upstream of codon 5 and 6) and β-TAG2 (whichbinds upstream of codon 30 and the IVS-1 donor site). Reaction productsare captured using streptavidin-coated paramagnetic particle boundbiotinylated capture primers (cap-tag-1 and cap-tag-2, respectively),that have 6 bases at the 5′ end that are complementary to the 5′ end ofβ-TAG1 and β-TAG2, respectively, and a portion which binds to auniversal primer.

FIG. 83 shows a mass spectrum of the PROBE products of a DNA sample fromone individual analyzed as described schematically in FIG. 82.

FIG. 84 shows a mass spectrum of the sequence bound to cap-tag-2.

FIG. 85 shows a mass spectrum obtained by using the β-TAG1 and β-TAG 2primers in one sequencing reaction using ddATP for termination and thensorting according to the method depicted in FIG. 82.

FIG. 86 shows a mass spectrum obtained by using the β-TAG1 and β-TAG2primers in one sequencing reaction using ddCTP for termination and thensorting according to the method depicted in FIG. 82.

FIG. 87A shows the wildtype sequence of a fragment of the chemokinereceptor CKR-5 gene with primers (SEQ ID NO: 73) used for amplification(SEQ ID NOs: 277, 292 and 293). In FIG. 87B, the wildtype strands aredepicted with and without an added Adenosine, their length and molecularmasses are indicated (SEQ ID NOs: 277, 296 and 297). FIG. 87C indicatesthe same for the 32 bp deletion (SEQ ID NO: 299). FIG. 87D shows thePROBE products for the wildtype gene (SEQ ID NO: 300) and FIG. 87E showsthe mutated allele (SEQ ID NO: 301).

FIG. 88 shows the amplification products of different unrelatedindividuals as analyzed by native polyacrylamide gel electrophoreses(15%) and silver stain. The band corresponding to a wildtype CKR-5 runsat 75 bp and the band from the gene with the deletion at 43 bp. Bandsbigger than 75 bp are due to unspecific amplification.

FIG. 89A shows a spectrograph of DNA derived from a heterozygousindividual: the peak with a mass of 23319 Da corresponds to the wildtypeCKR-5 and the peaks with masses of 13137 Da and 13451 Da to the deletionallele with and without an extra Adenosine, respectively. FIG. 89B showsa spectrograph of DNA obtained from the same individual as in FIG. 89A,but the DNA was treated with T4 DNA polymerase to remove the addedAdenosine. FIGS. 89C and 89D are spectrographs derived from homozygousindividuals and in FIG. 89D, the Adenosine has been removed. All peakswith masses lower than 13000 Da are due to multiple charged molecules.

FIG. 90A shows the mass spectrum of the results of a PROBE reactionperformed on DNA obtained from a heterozygous individual. FIG. 90B showsa mass spectrum of the results of a PROBE reaction on a homozygousindividual. The peaks with masses of 6604 Da and 6607 Da, respectivelycorrespond to the wildtype allele, and the peak with a mass of 6275 Dato the deletion allele. The primer is detected with a mass of 5673 and5676 Da, respectively.

FIG. 91 shows a MALDI-TOF MS spectra of a thermocycling primer OligoBase Extension (tc-PROBE) reaction as described in Example 24 usingthree different templates and 5 different PROBE primers simultaneouslyin one reaction.

FIG. 92 schematically depicts a single tube process for amplifying andsequencing exons 5-8 of the p53 gene as described in Example 25. Themass spectrum is the A reaction of FIG. 93.

FIG. 93 shows a superposition plot of four separate reactions forsequencing a portion (SEQ ID NO: 318) of exon 7 of the p53 gene asdescribed in Example 25.

FIG. 94 shows the mass spectrum obtained from the A reaction forsequencing a portion (SEQ ID NO: 319) of exon 7 of the p53 gene asdescribed in Example 25.

FIG. 95 shows the mass spectrum of a p53 sequencing ladder (set forth inSEQ ID NO: 320) for which 5 nL of each reaction were transferred towells of a chip and measured by MALDI-TOF.

FIG. 96A shows a MALDI-TOF mass spectra of a synthetic 50-mer (15.34kDa) mixed with 27-mer_(nc) (non-complementary, 8.30 kDa).

FIG. 96B shows a MALDI-TOF mass spectra of a synthetic 50-mer (15.34 kDa) mixed with a 27-mer_(c) (complementary, 8.34 kDa). The finalconcentration of each oligonucleotide was 10 μM. The signal at 23.68 kDain FIG. 96B corresponds to WC-specific dsDNA.

FIG. 97A shows a MALDI-TOF mass spectrum of CfoI/RsaI digest products ofa region of exon 4 of the apolipoprotein E gene (ε3 genotype), usingsample preparation as in FIG. 96.

FIG. 97B is the same as FIG. 97A, except with samples prepared forMALDI-TOF analysis at 4° C.

FIG. 98 shows a MALDI-TOF mass spectrum of CfoI/RsaI simultaneouslydouble digest products of a 252 base pair region of exon 4 of theapolipoprotein E gene (e4 genotype), with samples prepared at 4° C.

FIG. 99 shows the mass spectra obtained on a small population study of15 patients with a 16 element array of diagnostic products transferredto a MALDI target using a pintool microdispenser.

FIG. 100 is a MALDI mass spectrum of an aliquot sampled after a T₁digest of a synthetic 20-mer RNA (SEQ ID NO: 63).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. Where permitted the subject matterof each of the co-pending patent applications and the patent is hereinincorporated in its entirety.

As used herein, the term “biological sample” refers to any materialobtained from any living source (e.g., human, animal, plant, bacteria,fungi, protist, virus). For purposes herein, the biological sample willtypically contain a nucleic acid molecule. Examples of appropriatebiological samples include, but are not limited to: solid materials(e.g., tissue, cell pellets, biopsies) and biological fluids (e.g.,urine, blood, saliva, amniotic fluid, mouth wash, cerebral spinal fluidand other body fluids).

As used herein, the phrases “chain-elongating nucleotides” and“chain-terminating nucleotides” are used in accordance with their artrecognized meaning. For example, for DNA, chain-elongating nucleotidesinclude 2′deoxyribonucleotides (e.g., dATP, dCTP, dGTP and dTTP) andchain-terminating nucleotides include 2′,3′-dideoxyribonucleotides(e.g., ddATP, ddCTP, ddGTP, ddTTP). For RNA, chain-elongatingnucleotides include ribonucleotides (e.g., ATJP, CTP, GTP and UTP) andchain-terminating nucleotides include 3′-deoxyribonucleotides (e.g.,3′dA, 3′dC, 3′dG and 3′dU). A complete set of chain elongatingnucleotides refers to dATP, dCTP, dGTP and dTTP. The term “nucleotide”is also well known in the art.

As used herein, nucleotides include nucleoside mono-, di-, andtriphosphates. Nucleotides also include modified nucleotides such asphosphorothioate nucleotides and deazapurine nucleotides. A complete setof chain-elongating nucleotides refers to four different nucleotidesthat can hybridize to each of the four different bases comprising theDNA template.

As used herein, the superscript 0-i designates i+1 mass differentiatednucleotides, primers or tags. In some instances, the superscript 0 candesignate an unmodified species of a particular reactant, and thesuperscript i can designate the i-th mass-modified species of thatreactant. If, for example, more than one species of nucleic acids are tobe concurrently detected, then i+1 different mass-modified detectoroligonucleotides (D⁰, D¹, . . . D^(i)) can be used to distinguish eachspecies of mass modified detector oligonucleotides (D) from the othersby mass spectrometry.

As used herein, “multiplexing” refers to the simultaneously detection ofmore than one analyte, such as more than one (mutated) loci on aparticular captured nucleic acid fragment (on one spot of an array).

As used herein, the term “nucleic acid” refers to single-stranded and/ordouble-stranded polynucleotides such as deoxyribonucleic acid (DNA), andribonucleic acid (RNA) as well as analogs or derivatives of either RNAor DNA. Also included in the term “nucleic acid” are analogs of nucleicacids such as peptide nucleic acid (PNA), phosphorothioate DNA, andother such analogs and derivatives.

As used herein, the term “conjugated” refers stable attachment,preferably ionic or covalent attachment. Among preferred conjugationmeans are: streptavidin- or avidin-to biotin interaction; hydrophobicinteraction; magnetic interaction (e.g., using functionalized magneticbeads, such as DYNABEADS, which are streptavidin-coated magnetic beadssold by Dynal, Inc. Great Neck, N.Y. and Oslo Norway); polarinteractions, such as “wetting” associations between two polar surfacesor between oligo/polyethylene glycol; formation of a covalent bond, suchas an amide bond, disulfide bond, thioether bond, or via crosslinkingagents; and via an acid-labile or photocleavable linker.

As used herein equivalent, when referring to two sequences of nucleicacids means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When “equivalent” is used inreferring to two proteins or peptides, it means that the two proteins orpeptides have substantially the same amino acid sequence with onlyconservative amino acid substitutions that do not substantially alterthe activity or function of the protein or peptide. When “equivalent”refers to a property, the property does not need to be present to thesame extent (e.g., two peptides can exhibit different rates of the sametype of enzymatic activity), but the activities are preferablysubstantially the same. “Complementary,” when referring to twonucleotide sequences, means that the two sequences of nucleotides arecapable of hybridizing, preferably with less than 25%, more preferablywith less than 15%, even more preferably with less than 5%, mostpreferably with no mismatches between opposed nucleotides. Preferablythe two molecules will hybridize under conditions of high stringency.

As used herein: stringency of hybridization in determining percentagemismatch are those conditions understood by those of skill in the artand typically are substantially equivalent to the following:

1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C.

2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C.

3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C.

It is understood that equivalent stringencies may be achieved usingalternative buffers, salts and temperatures.

As used herein, a primer when set forth in the claims refers to a primersuitable for mass spectrometric methods requiring immobilizing,hybridizing, strand displacement, sequencing mass spectrometry refers toa nucleic acid must be of low enough mass, typically about 70nucleotides or less than 70, and of sufficient size to be useful in themass spectrometric methods described herein that rely on massspectrometric detection. These methods include primers for detection andsequencing of nucleic acids, which require a sufficient numbernucleotides to from a stable duplex, typically about 6-30, preferablyabout 10-25, more preferably about 12-20. Thus, for purposes herein aprimer will be a sequence of nucleotides comprising about 6-70, morepreferably a 12-70, more preferably greater than about 14 to an upperlimit of 70, depending upon sequence and application of the primer. Theprimers herein, for example for mutational analyses, are selected to beupstream of loci useful for diagnosis such that when performing usingsequencing up to or through the site of interest, the resulting fragmentis of a mass that sufficient and not too large to be detected by massspectrometry. For mass spectrometric methods, mass tags or modifier arepreferably included at the 5′-end, and the primer is otherwiseunlabeled.

As used herein, “conditioning” of a nucleic acid refers to modificationof the phosphodiester backbone of the nucleic acid molecule (e.g.,cation exchange) for the purpose of eliminating peak broadening due to aheterogeneity in the cations bound per nucleotide unit. Contacting anucleic acid molecule with an alkylating agent such as alkyliodide,iodoacetamide, β-iodoethanol, or 2,3-epoxy-1-propanol, the monothiophosphodiester bonds of a nucleic acid molecule can be transformed intoa phosphotriester bond. Likewise, phosphodiester bonds may betransformed to uncharged derivatives employing trialkylsilyl chlorides.Further conditioning involves incorporating nucleotides that reducesensitivity for depurination (fragmentation during MS) e.g., a purineanalog such as N7- or N9-deazapurine nucleotides, or RNA building blocksor using oligonucleotide triesters or incorporating phosphorothioatefunctions that are alkylated or employing oligonucleotide mimetics suchas peptide nucleic acid (PNA).

As used herein, substrate refers to an insoluble support onto which asample is deposited according to the materials described herein.Examples of appropriate substrates include beads (e.g., silica gel,controlled pore glass, magnetic, agarose gel and crosslinked dextroses(i.e. Sepharose and Sephadex, cellulose and other materials known bythose of skill in the art to serve as solid support matrices. Forexample substrates may be formed from any or combinations of: silicagel, glass, magnet, polystyrene/1% divinylbenzene resins, such as Wangresins, which are Fmoc-amino acid-4-(hydroxymethyl)-phenoxymethylcopoly(styrene-1% divinylbenzene (DVD)) resin, chlorotrityl(2-chlorotritylchloride copolystyrene-DVB resin) resin, Merrifield(chloromethylated copolystyrene-DVB) resin metal, plastic, cellulose,cross-linked dextrans, such as those sold under the tradename Sephadex(Pharmacia) and agarose gel, such as gels sold under the tradenameSepharose (Pharmacia), which is a hydrogen bonded polysaccharide-typeagarose gel, and other such resins and solid phase supports known tothose of skill in the art. The support matrices may be in any shape orform, including, but not limited to: capillaries, flat supports such asglass fiber filters, glass surfaces, metal surfaces (steel, gold,silver, aluminum, copper and silicon), plastic materials includingmultiwell plates or membranes (e.g., of polyethylene, polypropylene,polyamide, polyvinylidenedifluoride), pins (e.g., arrays of pinssuitable for combinatorial synthesis or analysis or beads in pits offlat surfaces such as wafers (e.g., silicon wafers) with or withoutplates, and beads.

As used herein, a selectively cleavable linker is a linker that iscleaved under selected conditions, such as a photocleavable linker, achemically cleavable linker and an enzymatically cleavable linker (i.e.,a restriction endonuclease site or a ribonucleotide/RNase digestion).The linker is interposed between the support and immobilized DNA.

Isolation of Nucleic Acids Molecules

Nucleic acid molecules can be isolated from a particular biologicalsample using any of a number of procedures, which are well-known in theart, the particular isolation procedure chosen being appropriate for theparticular biological sample. For example, freeze-thaw and alkalinelysis procedures can be useful for obtaining nucleic acid molecules fromsolid materials; heat and alkaline lysis procedures can be useful forobtaining nucleic acid molecules from urine; and proteinase K extractioncan be used to obtain nucleic acid from blood (see, e.g., Rolff et al(1994) PCR: Clinical Diagnostics and Research, Springer).

To obtain an appropriate quantity of a nucleic acid molecules on whichto perform mass spectrometry, amplification may be necessary. Examplesof appropriate amplification procedures for use herein include: cloning(Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, 1989), polymerase chain reaction (PCR) (C. R.Newton and A. Graham, PCR, BIOS Publishers, 1994), ligase chain reaction(LCR) (see, e.g., Weidmann et al. (1994) PCR Methods Appl. Vol. 3, Pp.57-64; F. Barany (1991) Proc. Natl. Acad. Sci. U.S.A. 88: 189-93),strand displacement amplification (SDA) (see, e.g., Walker et al. (1994)Nucleic Acids Res. 22: 2670-77) and variations such as RT-PCR (see,e.g., Higuchi et al. (1993) Bio/Technology 11: 1026-1030),allele-specific amplification (ASA) and transcription based processes.

Immobilization of Nucleic Acid Molecules to Solid Supports

To facilitate mass spectrometric analysis, a nucleic acid moleculecontaining a nucleic acid sequence to be detected can be immobilized toan insoluble (i.e., a solid) support. Examples of appropriate solidsupports include beads (e.g., silica gel, controlled pore glass,magnetic, Sephadex/Sepharose, cellulose), capillaries, flat supportssuch as glass fiber filters, glass surfaces, metal surfaces (steel,gold, silver, aluminum, copper and silicon), plastic materials includingmultiwell plates or membranes (e.g. it would have been obvious to one ofordinary skill in the art to have, of polyethylene, polypropylene,polyamide, polyvinylidenedifluoride), pins (e.g., arrays of pinssuitable for combinatorial synthesis or analysis or beads in pits offlat surfaces such as wafers (e.g., silicon wafers) with or withoutfilter plates.

Samples containing target nucleic acids can be transferred to solidsupports by any of a variety of methods known to those of skill in theart. For example, nucleic acid samples can be transferred to individualwells of a substrate, e.g., silicon chip, manually or using a pintoolmicrodispenser apparatus as described herein. Alternatively, apiezoelectric pipette apparatus can be used to transfer small nanolitersamples to a substrate permitting the performance of high throughputminiaturized diagnostics on a chip.

Immobilization can be accomplished, for example, based on hybridizationbetween a capture nucleic acid sequence, which has already beenimmobilized to the support and a complementary nucleic acid sequence,which is also contained within the nucleic acid molecule containing thenucleic acid sequence to be detected (FIG. 1A). So that hybridizationbetween the complementary nucleic acid molecules is not hindered by thesupport, the capture nucleic acid can include an e.g., spacer region ofat least about five nucleotides in length between the solid support andthe capture nucleic acid sequence. The duplex formed will be cleavedunder the influence of the laser pulse and desorption can be initiated.The solid support-bound nucleic acid molecule can be presented throughnatural oligoribo- or oligodeoxyribonucleotide as well as analogs (e.g.,thio-modified phosphodiester or phosphotriester backbone) or employingoligonucleotide mimetics such as PNA analogs (see, e.g., Nielsen et al.,Science 254: 1497 (1991)) which render the base sequence lesssusceptible to enzymatic degradation and -bound capture base sequence.

Linkers

A target detection site can be directly linked to a solid support via areversible or irreversible bond between an appropriate functionality(L′) on the target nucleic acid molecule (T) and an appropriatefunctionality (L) on the capture molecule (FIG. 1B). A reversiblelinkage can be such that it is cleaved under the conditions of massspectrometry (i.e., a photocleavable bond such as a charge transfercomplex or a labile bond being formed between relatively stable organicradicals).

Photocleavable linkers are linkers that are cleaved upon exposure tolight (see, e.g., Goldmacher et al. (1992) Bioconj. Chem. 3: 104-107),thereby releasing the targeted agent upon exposure to light.Photocleavable linkers that are cleaved upon exposure to light are known(see, e.g., Hazum et al. (1981) in Pept., Proc. Eur. Pept. Symp., 16th,Brunfeldt, K (Ed), pp. 105-110, which describes the use of a nitrobenzylgroup as a photocleavable protective group for cysteine; Yen et al.(1989) Makromol. Chem. 190: 69-82, which describes water solublephotocleavable copolymers, including hydroxypropylmethacrylamidecopolymer, glycine copolymer, fluorescein copolymer and methylrhodaminecopolymer; Goldmacher et al. (1992) Bioconj. Chem. 3: 104-107, whichdescribes a cross-linker and reagent that undergoes photolyticdegradation upon exposure to near UV light (350 nm); and Senter et al.(1985) Photochem. Photobiol 42: 231-237, which describesnitrobenzyloxycarbonyl chloride cross linking reagents that producephotocleavable linkages), thereby releasing the targeted agent uponexposure to light. In preferred embodiments, the nucleic acid isimmobilized using the photocleavable linker moiety that is cleavedduring mass spectrometry. Presently preferred photocleavable linkers areset forth in the EXAMPLES.

Furthermore, the linkage can be formed with L′ being a quaternaryammonium group, in which case, preferably, the surface of the solidsupport carries negative charges which repel the negatively chargednucleic acid backbone and thus facilitate the desorption required foranalysis by a mass spectrometer. Desorption can occur either by the heatcreated by the laser pulse and/or, depending on L,′ by specificabsorption of laser energy which is in resonance with the L′chromophore.

Thus, the L-L′ chemistry can be of a type of disulfide bond (chemicallycleavable, for example, by mercaptoethanol or dithioerythrol), abiotin/streptavidin system, a heterobifunctional derivative of a tritylether group (see, e.g., Köster et al. (1990) “A Versatile Acid-LabileLinker for Modification of Synthetic Biomolecules,” Tetrahedron Letters31: 7095) that can be cleaved under mildly acidic conditions as well asunder conditions of mass spectrometry, a levulinyl group cleavable underalmost neutral conditions with a hydrazinium/acetate buffer, anarginine-arginine or lysine-lysine bond cleavable by an endopeptidaseenzyme like trypsin or a pyrophosphate bond cleavable by apyrophosphatase, or a ribonucleotide bond in between theoligodeoxynucleotide sequence, which can be cleaved, for example, by aribonuclease or alkali.

The functionalities, L and L,′ can also form a charge transfer complexand thereby form the temporary L-L′ linkage. Since in many cases the“charge-transfer band” can be determined by UV/vis spectrometry (see,e.g., Organic Charge Transfer Complexes by R. Foster, Academic Press,1969), the laser energy can be tuned to the corresponding energy of thecharge-transfer wavelength and, thus, a specific desorption off thesolid support can be initiated. Those skilled in the art will recognizethat several combinations can serve this purpose and that the donorfunctionality can be either on the solid support or coupled to thenucleic acid molecule to be detected or vice versa.

In yet another approach, a reversible L-L′ linkage can be generated byhomolytically forming relatively stable radicals. Under the influence ofthe laser pulse, desorption (as discussed above) as well as ionizationwill take place at the radical position. Those skilled in the art willrecognize that other organic radicals can be selected and that, inrelation to the dissociation energies needed to homolytically cleave thebond between them, a corresponding laser wavelength can be selected (seee.g., Reactive Molecules by C. Wentrup, John Wiley & Sons, 1984).

An anchoring function L′ can also be incorporated into a targetcapturing sequence (TCS) by using appropriate primers during anamplification procedure, such as PCR (FIG. 4), LCR (FIG. 5) ortranscription amplification (FIG. 6A).

When performing exonuclease sequencing using MALDI-TOF MS, a singlestranded DNA molecule immobilized via its 5-end to a solid support isunilaterally degraded with a 3′-processive exonuclease and the molecularweight of the degraded nucleotide is determined sequentially. ReverseSanger sequencing reveals the nucleotide sequence of the immobilizedDNA. By adding a selectively cleavable linker, not only can the mass ofthe free nucleotides be determined but also, upon removal of thenucleotides by washing, the mass of the remaining fragment can bedetected by MALDI-TOF upon cleaving the DNA from the solid support.Using selectively cleavable linkers, such as the photocleavable andchemical cleavable linkers provided herein, this cleavage can beselected to occur during the ionization and volatizing steps ofMALDI-TOF. The same rationale applies for a 5′ immobilized strand of adouble stranded DNA that is degraded while in a duplex. Likewise, thisalso applies when using a 5′-processive exonuclease and the DNA isimmobilized through the 3′-end to the solid support.

As noted, at least three version of immobilization are contemplatedherein: 1) the target nucleic acid is amplified or obtained (the targetsequence or surrounding DNA sequence must be known to make primers toamplify or isolated); 2) the primer nucleic acid is immobilized to thesolid support and the target nucleic acid is hybridized thereto (this isfor detecting the presence of or sequencing a target sequence in asample); or 3) a double stranded DNA (amplified or isolated) isimmobilized through linkage to one predetermined strand, the DNA isdenatured to eliminate the duplex and then a high concentration of acomplementary primer or DNA with identity upstream from the target siteis added and a strand displacement occurs and the primer is hybridizedto the immobilized strand.

In the embodiments where the primer nucleic acid is immobilized on thesolid support and the target nucleic acid is hybridized thereto, theinclusion of the cleavable linker allows the primer DNA to beimmobilized at the 5′-end so that free 3′-OH is available for nucleicacid synthesis (extension) and the sequence of the “hybridized” targetDNA can be determined because the hybridized template can be removed bydenaturation and the extended DNA products cleaved from the solidsupport for MALDI-TOF MS. Similarly for 3), the immobilized DNA strandcan be elongated when hybridized to the template and cleaved from thesupport. Thus, Sanger sequencing and primer oligo base extension(PROBE), discussed below, extension reactions can be performed using animmobilized primer of a known, upstream DNA sequence complementary to aninvariable region of a target sequence. The nucleic acid from the personis obtained and the DNA sequence of a variable region (deletion,insertion, missense mutation that cause genetic predisposition ordiseases, or the presence of viral/bacterial or fungal DNA) not only isdetected, but the actual sequence and position of the mutation is alsodetermined.

In other cases, the target DNA must be immobilized and the primerannealed. This requires amplifying a larger DNA based on known sequenceand then sequencing the immobilized fragments (i.e., the extendedfragments are hybridized but not immobilized to the support as describedabove). In these cases, it is not desirable to include a linker becausethe MALDI-TOF spectrum is of the hybridized DNA; it is not necessary tocleave the immobilized template.

Any linker known to those of skill in the art for immobilizing nucleicacids to solid supports may be used herein to link the nucleic acid to asolid support. The preferred linkers herein are the selectivelycleavable linkers, particularly those exemplified herein. Other linkersinclude, acid cleavable linkers, such as bismaleimideothoxy propane,acid-labile trityl linkers.

Acid cleavable linkers, photocleavable and heat sensitive linkers mayalso be used, particularly where it may be necessary to cleave thetargeted agent to permit it to be more readily accessible to reaction.Acid cleavable linkers include, but are not limited to,bismaleimideothoxy propane; and adipic acid dihydrazide linkers (see,e.g., Fattom et al. (1992) Infection & Immun. 60: 584-589) and acidlabile transferrin conjugates that contain a sufficient portion oftransferrin to permit entry into the intracellular transferrin cyclingpathway (see, e.g., Welhöner et al. (1991) J. Biol. Chem. 266:4309-4314).

Photocleavable Linkers

Photocleavable linkers are provided. In particular, photocleavablelinkers as their phosphoramidite derivatives are provided for use insolid phase synthesis of oligonucleotides. The linkers containo-nitrobenzyl moieties and phosphate linkages which allow for completephotolytic cleavage of the conjugates within minutes upon UVirradiation. The UV wavelengths used are selected so that theirradiation will not damage the oligonucleotides and are preferablyabout 350-380 nm, more preferably 365 nm. The photocleavable linkersprovided herein possess comparable coupling efficiency as compared tocommonly used phosphoramidite monomers (see, Sinha et al. (1983)Tetrahedron Lett. 24: 5843-5846; Sinha et al. (1984) Nucleic Acids Res.12: 4539-4557; Beaucage et al. (1993) Tetrahedron 49: 6123-6194; andMatteucci et al. (1981) J. Am. Chem. Soc. 103: 3185-3191).

In one embodiment, the photocleavable linkers have formula I:

where R²⁰ is ω-(4,4′-dimethoxytrityloxy)alkyl or ω-hydroxyalkyl; R²¹ isselected from hydrogen, alkyl, aryl, alkoxycarbonyl, aryloxycarbonyl andcarboxy; R²² is hydrogen or (dialkylamino)(ω-cyanoalkoxy)P—; t is 0-3;and R⁵⁰ is alkyl, alkoxy, aryl or aryloxy.

In a preferred embodiment, the photocleavable linkers have formula II:

where R²⁰ is ω-(4,4′-dimethoxytrityloxy)alkyl, ω-hydroxyalkyl or alkyl;R²¹ is selected from hydrogen, alkyl, aryl, alkoxycarbonyl,aryloxycarbonyl and carboxy; R²² is hydrogen or(dialkylamino)(ω-cyanoalkoxy)P—; and X²⁰ is hydrogen, alkyl or OR²⁰.

In particularly preferred embodiments, R²⁰ is3-(4,4′-dimethoxytrityloxy)propyl, 3-hydroxypropyl or methyl; R²¹ isselected from hydrogen, methyl and carboxy; R²² is hydrogen or(diisopropylamino)(2-cyanoethoxy)P—; and X²⁰ is hydrogen, methyl orOR²⁰. In a more preferred embodiment, R²⁰ is3-(4,4′-dimethoxytrityloxy)propyl; R²¹ is methyl; R²² is(diisopropylamino)(2-cyanoethoxy)P—; and X²⁰ is hydrogen. In anothermore preferred embodiment, R²⁰ is methyl; R²¹ is methyl; R²² is(diisopropylamino)(2-cyanoethoxy)P—; and X²⁰ is3-(4,4′-dimethoxytrityloxy)propoxy.

In another embodiment, the photocleavable linkers have formula III:

where R²³ is hydrogen or (dialkylamino)(ω-cyanoalkoxy)P—; and R²⁴ isselected from ω-hydroxyalkoxy, ω-(4,4′-dimethoxytrityloxy)alkoxy,ω-hydroxyalkyl and ω-(4,4′-dimethoxytrityloxy)alkyl, and isunsubstituted or substituted on the alkyl or alkoxy chain with one ormore alkyl groups; r and s are each independently 0-4; and R⁵⁰ is alkyl,alkoxy, aryl or aryloxy. In certain embodiments, R²⁴ is ω-hydroxyalkylor ω-(4,4′-dimethoxytrityloxy)alkyl, and is substituted on the alkylchain with a methyl group.

In preferred embodiments, R²³ is hydrogen or(diisopropylamino)(2-cyanoethoxy)P—; and R²⁴ is selected from3-hydroxypropoxy, 3-(4,4′-dimethoxytrityloxy)propoxy, 4-hydroxybutyl,3-hydroxy-1-propyl, 1-hydroxy-2-propyl, 3-hydroxy-2-methyl-1-propyl,2-hydroxyethyl, hydroxymethyl, 4-(4,4′-dimethoxytrityloxy)butyl,3-(4,4′-dimethoxytrityloxy)-1-propyl, 2-(4,4′-dimethoxytrityloxy)ethyl,1-(4,4′-dimethoxytrityloxy)-2-propyl,3-(4,4′-dimethoxytrityloxy)-2-methyl-1-propyl and4,4′-dimethoxytrityloxymethyl.

In more preferred embodiments, R²³ is(diisopropylamino)(2-cyanoethoxy)P—; r and s are 0; and R²⁴ is selectedfrom 3-(4,4′-dimethoxytrityloxy)propoxy,4-(4,4′-dimethoxytrityloxy)butyl, 3-(4,4′-dimethoxytrityloxy)propyl,2-(4,4′-dimethoxytrityloxy)ethyl, 1-(4,4′-dimethoxytrityloxy)-2-propyl,3-(4,4′-dimethoxytrityloxy)-2-methyl-1-propyl and4,4′-dimethyoxytrityloxymethyl. R²⁴ is most preferably3-(4,4′-dimethoxytrityloxy)propoxy.

Preparation of the Photocleavable Linkers

A. Preparation of Photocleavable Linkers of Formulae I or II

Photocleavable linkers of formulae I or II may be prepared by themethods described below, by minor modification of the methods bychoosing the appropriate starting materials or by any other methodsknown to those of skill in the art. Detailed procedures for thesynthesis of photocleavable linkers of formula II are provided in theExamples.

In the photocleavable linkers of formula II where X²⁰ is hydrogen, thelinkers may be prepared in the following manner. Alkylation of5-hydroxy-2-nitrobenzaldehyde with an ω-hydroxyalkyl halide, e.g.,3-hydroxypropyl bromide, followed by protection of the resulting alcoholas, e.g., a silyl ether, provides a5-(ω-silyloxyalkoxy)-2-nitrobenzaldehyde. Addition of an organometallicto the aldehyde affords a benzylic alcohol. Organometallics which may beused include trialkylaluminums (for linkers where R²¹ is alkyl), such astrimethylaluminum, borohydrides (for linkers where R²¹ is hydrogen),such as sodium borohydride, or metal cyanides (for linkers where R²¹ iscarboxy or alkoxycarbonyl), such as potassium cyanide. In the case ofthe metal cyanides, the product of the reaction, a cyanohydrin, wouldthen be hydrolyzed under either acidic or basic conditions in thepresence of either water or an alcohol to afford the compounds ofinterest.

The silyl group of the side chain of the resulting benzylic alcohols maythen be exchanged for a 4,4′-dimethoxytrityl group by desilylation with,e.g., tetrabutylammonium fluoride, to give the corresponding alcohol,followed by reaction with 4,4′-dimethoxytrityl chloride. Reaction with,e.g., 2-cyanoethyl diisopropylchlorophosphoramidite affords the linkerswhere R²² is (dialkylamino)(ω-cyanoalkoxy)P—.

A specific example of a synthesis of a photocleavable linker of formulaII is shown in the following scheme, which also demonstrates use of thelinker in oligonucleotide synthesis. This scheme is intended to beillustrative only and in no way limits the scope of the invention.Experimental details of these synthetic transformations are provided inthe Examples.

Synthesis of the linkers of formula II where X²⁰ is OR²⁰,3,4-dihydroxyacetophenone is protected selectively at the 4-hydroxyl byreaction with, e.g., potassium carbonate and a silyl chloride. Benzoateesters, propiophenones, butyrophenones, etc. may be used in place of theacetophenone. The resulting 4-silyloxy-3-hydroxyacetophenone is thenalkylated at the with an alkyl halide (for linkers where R²⁰ is alkyl)at the 3-hydroxyl and desilylated with, e.g., tetrabutylammoniumfluoride to afford a 3-alkoxy-4-hydroxyacetophenone. This compound isthen alkylated at the 4-hydroxyl by reaction with an ω-hydroxyalkylhalide, e.g., 3-hydroxypropyl bromide, to give a4-(ω-hydroxyalkoxy)-3-alkoxyacetophenone. The side chain alcohol is thenprotected as an ester, e.g., an acetate. This compound is then nitratedat the 5-position with, e.g., concentrated nitric acid to provide thecorresponding 2-nitroacetophenones. Saponification of the side chainester with, e.g., potassium carbonate, and reduction of the ketone with,e.g., sodium borohydride, in either order gives a2-nitro-4-(ω-hydroxyalkoxy)-5-alkoxybenzylic alcohol.

Selective protection of the side chain alcohol as the corresponding4,4′-dimethoxytrityl ether is then accomplished by reaction with4,4′-dimethoxytrityl chloride. Further reaction with, e.g., 2-cyanoethyldiisopropylchlorophosphoramidite affords the linkers where R²² is(dialkylamino)(ω-cyanoalkoxy)P—.

A specific example of the synthesis of a photocleavable linker offormula II is shown the following scheme. This scheme is intended to beillustrative only and in no way limit the scope of the invention.Detailed experimental procedures for the transformations shown are foundin the Examples.

B. Preparation of Photocleavable Linkers of Formula III

Photocleavable linkers of formula III may be prepared by the methodsdescribed below, by minor modification of the methods by choosingappropriate starting materials, or by other methods known to those ofskill in the art.

In general, photocleavable linkers of formula III are prepared fromω-hydroxyalkyl- or alkoxyaryl compounds, in particular ω-hydroxy-alkylor alkoxy-benzenes. These compounds are commercially available, or maybe prepared from an ω-hydroxyalkyl halide (e.g., 3-hydroxypropylbromide) and either phenyllithium (for the ω-hydroxyalkylbenzenes) or

phenol (for the ω-hydroxyalkoxybenzenes). Acylation of the ω-hydroxylgroup (e.g., as an acetate ester) followed by Friedel-Crafts acylationof the aromatic ring with 2-nitrobenzoyl chloride provides a4-(ω-acetoxy-alkyl or alkoxy)-2-nitrobenzophenone. Reduction of theketone with, e.g., sodium borohydride, and saponification of the sidechain ester are performed in either order to afford a2-nitrophenyl-4-(hydroxy-alkyl or alkoxy)phenylmethanol. Protection ofthe terminal hydroxyl group as the corresponding 4,4′-dimethoxytritylether is achieved by reaction with 4,4′-dimethoxytrityl chloride. Thebenzylic hydroxyl group is then reacted with, e.g., 2-cyanoethyldiisopropylchlorophosphoramidite to afford linkers of formula II whereR²³ is (dialkylamino)(ω-cyanoalkoxy)P—.

Other photocleavable linkers of formula III may be prepared bysubstituting 2-phenyl-1-propanol or 2-phenylmethyl-1-propanol for theω-hydroxy-alkyl or alkoxy-benzenes in the above synthesis. Thesecompounds are commercially available, but may also be prepared byreaction of, e.g., phenylmagnesium bromide or benzylmagnesium bromide,with the requisite oxirane (i.e., propylene oxide) in the presence ofcatalytic cuprous ion.

Chemically Cleavable Linkers

A variety of chemically cleavable linkers may be used to introduce acleavable bond between the immobilized nucleic acid and the solidsupport. Acid-labile linkers are presently preferred chemicallycleavable linkers for mass spectrometry, especially MALDI-TOF MS,because the acid labile bond is cleaved during conditioning of thenucleic acid upon addition of the 3-HPA matrix solution. The acid labilebond can be introduced as a separate linker group, e.g., the acid labiletrityl groups (see FIG. 68; Example 16) or may be incorporated in asynthetic nucleic acid linker by introducing one or more silylinternucleoside bridges using diisopropylsilyl, thereby formingdiisopropylsilyl-linked oligonucleotide analogs. The diisopropylsilylbridge replaces the phoshodiester bond in the DNA backbone and undermildly acidic conditions, such as 1.5% trifluoroacetic acid (TFA) or3-HPA/1% TFA MALDI-TOF matrix solution, results in the introduction ofone or more intra-strand breaks in the DNA molecule. Methods for thepreparation of diisopropylsilyl-linked oligonucleotide precursors andanalogs are known to those of skill in the art (see e.g., Saha et al.(1993) J. Org. Chem. 58: 7827-7831). These oligonucleotide analogs maybe readily prepared using solid state oligonucleotide synthesis methodsusing diisopropylsilyl derivatized deoxyribonucleosides.

Nucleic Acid Conditioning

Prior to mass spectrometric analysis, it may be useful to “condition”nucleic acid molecules, for example to decrease the laser energyrequired for volatilization and/or to minimize fragmentation.Conditioning is preferably performed while a target detection site isimmobilized. An example of conditioning is modification of thephosphodiester backbone of the nucleic acid molecule (e.g., cationexchange), which can be useful for eliminating peak broadening due to aheterogeneity in the cations bound per nucleotide unit. Contacting anucleic acid molecule with an alkylating agent such as alkyliodide,iodoacetamide, β-iodoethanol, or 2,3-epoxy-1-propanol, the monothiophosphodiester bonds of a nucleic acid molecule can be transformed intoa phosphotriester bond. Likewise, phosphodiester bonds may betransformed to uncharged derivatives employing trialkylsilyl chlorides.Further conditioning involves incorporating nucleotides that reducesensitivity for depurination (fragmentation during MS) e.g., a purineanalog such as N7- or N9-deazapurine nucleotides, or RNA building blocksor using oligonucleotide triesters or incorporating phosphorothioatefunctions which are alkylated or employing oligonucleotide mimetics suchas PNA.

Multiplex Reactions

For certain applications, it may be useful to simultaneously detect morethan one (mutated) loci on a particular captured nucleic acid fragment(on one spot of an array) or it may be useful to perform parallelprocessing by using oligonucleotide or oligonucleotide mimetic arrays onvarious solid supports. “Multiplexing” can be achieved by severaldifferent methodologies. For example, several mutations can besimultaneously detected on one target sequence by employingcorresponding detector (probe) molecules (e.g., oligonucleotides oroligonucleotide mimetics). The molecular weight differences between thedetector oligonucleotides D1, D2 and D3 must be large enough so thatsimultaneous detection (multiplexing) is possible. This can be achievedeither by the sequence itself (composition or length) or by theintroduction of mass-modifying functionalities M1-M3 into the detectoroligonucleotide (see FIG. 2).

Mass Modification of Nucleic Acids

Mass modifying moieties can be attached, for instance, to either the5′-end of the oligonucleotide (M¹), to the nucleobase (or bases) (M²,M⁷), to the phosphate backbone (M³), and to the 2′-position of thenucleoside (nucleosides) (M⁴, M⁶) and/or to the terminal 3′-position(M⁵). Examples of mass modifying moieties include, for example, ahalogen, an azido, or of the type, XR, wherein X is a linking group andR is a mass-modifying functionality. The mass-modifying functionalitycan thus be used to introduce defined mass increments into theoligonucleotide molecule.

The mass-modifying functionality can be located at different positionswithin the nucleotide moiety (see, e.g., U.S. Pat. No. 5,547,835 andInternational PCT application No. WO 94/21822). For example, themass-modifying moiety, M, can be attached either to the nucleobase, M²(in case of the c⁷-deazanucleosides also to C-7, M⁷), to thetriphosphate group at the alpha phosphate, M³, or to the 2′-position ofthe sugar ring of the nucleoside triphosphate, M⁴ and M⁶. Modificationsintroduced at the phosphodiester bond (M4), such as with alpha-thionucleoside triphosphates, have the advantage that these modifications donot interfere with accurate Watson-Crick base-pairing and additionallyallow for the one-step post-synthetic site-specific modification of thecomplete nucleic acid molecule e.g., via alkylation reactions (see,e.g., Nakamaye et al. (1988) Nucl. Acids Res. 16: 9947-59). Particularlypreferred mass-modifying functionalities are boron-modified nucleicacids since they are better incorporated into nucleic acids bypolymerases (see, e.g., Porter et al. (1995) Biochemistry 34:11963-11969; Hasan et al. (1996) Nucleic Acids Res. 24: 2150-2157; Li etal. (1995) Nucl. Acids Res. 23: 4495-4501).

Furthermore, the mass-modifying functionality can be added so as toaffect chain termination, such as by attaching it to the 3′-position ofthe sugar ring in the nucleoside triphosphate, M⁵. For those skilled inthe art, it is clear that many combinations can be used in the methodsprovided herein. In the same way, those skilled in the art willrecognize that chain-elongating nucleoside triphosphates can also bemass-modified in a similar fashion with numerous variations andcombinations in functionality and attachment positions.

Without being bound to any particular theory, the mass-modification, M,can be introduced for X in XR as well as using oligo-/polyethyleneglycol derivatives for R. The mass-modifying increment in this case is44, i.e. five different mass-modified species can be generated by justchanging m from 0 to 4 thus adding mass units of 45 (m=0), 89 (m=1), 133(m=2), 177 (m=3) and 221 (m=4) to the nucleic acid molecule (e.g.,detector oligonucleotide (D) or the nucleoside triphosphates (FIG.6(C)), respectively). The oligo/polyethylene glycols can also bemonoalkylated by a lower alkyl such as methyl, ethyl, propyl, isopropyl,t-butyl and the like. A selection of linking functionalities, X, arealso illustrated. Other chemistries can be used in the mass-modifiedcompounds (see, e.g., those described in Oligonucleotides and Analogues,A Practical Approach, F. Eckstein, editor, IRL Press, Oxford, 1991).

In yet another embodiment, various mass-modifying functionalities, R,other than oligo/polyethylene glycols, can be selected and attached viaappropriate linking chemistries, X. A simple mass-modification can beachieved by substituting H for halogens like F, Cl, Br and/or I, orpseudohalogens such as CN, SCN, NCS, or by using different alkyl, arylor aralkyl moieties such as methyl, ethyl, propyl, isopropyl, t-butyl,hexyl, phenyl, substituted phenyl, benzyl, or functional groups such asCH₂F, CHF₂, CF₃, Si(CH₃)₃, Si(CH₃)₂(C₂H₅), Si(CH₃)(C₂H₅)₂, Si(C₂H₅)₃.Yet another mass-modification can be obtained by attaching homo- orheteropeptides through the nucleic acid molecule (e.g., detector (D)) ornucleoside triphosphates. One example. useful in generatingmass-modified species with a mass increment of 57. is the attachment ofoligoglycines, e.g., mass-modifications of 74 (r=l, m=0), 131 (r=l,m=1), 188 (r=l, m=2), 245 (r=l, m=3) are achieved. Simple oligoamidesalso can be used, e.g., mass-modifications of 74 (r=l, m=0), 88 (r=2,m=0), 102 (r=3, m=0), 116 (r=4, m=0), etc. are obtainable. Variations inadditions to those set forth herein will be apparent to the skilledartisan.

Different mass-modified detector oligonucleotides can be used tosimultaneously detect all possible variants/mutants simultaneously (FIG.6B). Alternatively, all four base permutations at the site of a mutationcan be detected by designing and positioning a detector oligonucleotide,so that it serves as a primer for a DNA/RNA polymerase with varyingcombinations of elongating and terminating nucleoside triphosphates(FIG. 6C). For example, mass modifications also can be incorporatedduring the amplification process.

FIG. 3 shows a different multiplex detection format, in whichdifferentiation is accomplished by employing different specific capturesequences which are position-specifically immobilized on a flat surface(e.g., a ‘chip array’). If different target sequences T1-Tn are present,their target capture sites TCS1-TCSn will specifically interact withcomplementary immobilized capture sequences C1-Cn. Detection is achievedby employing appropriately mass differentiated detector oligonucleotidesD1-Dn, which are mass modifying functionalities M1-Mn.

Mass Spectrometric Methods for Sequencing DNA

Amenable mass spectrometric formats for use herein include theionization (I) techniques, such as matrix assisted laser desorptionionization (MALDI), electrospray (ESI) (e.g., continuous or pulsed); andrelated methods (e.g., Ionspray, Thermospray, Fast Atomic Bombardment),and massive cluster impact (MCI); these ion sources can be matched withdetection formats including lin-linear fields) time-of-flight (TOF),single or multiple quadrupole, single or multiple magnetic sector,Fourier transform ion cyclotron resonance (FTICR), ion trap, orcombinations of these to give a hybrid detector (e.g., ion trap-time offlight). For ionization, numerous matrix/wavelength combinationsincluding frozen analyte preparation (MALDI) or solvent combinations(ESI) can be employed.

Since a normal DNA molecule includes four nucleotide units (A, T, C, G),and the mass of each of these is unique (monoisotopic masses 313.06,304.05, 289.05, 329.05 Da, respectively), an accurate mass determinationcan define or constrain the possible base compositions of that DNA. Onlyabove 4900 Da does each unit molecular weight have at least oneallowable composition; among all 5-mers there is only one non-uniquenominal molecular weight, among 8-mers, 20. For these and largeroligonucleotides, such mass overlaps can be resolved with the ˜1/10⁵(˜10 part per million, ppm) mass accuracy available with high resolutionFTICR MS. For the 25-mer A₅T₂₀, the 20 composition degeneracies whenmeasured at ±0.5 Da is reduced to three (A₅T₂₀, T₄C₁₂G₉, AT₃C₄G₁₆) whenmeasured with 2 ppm accuracy. Given composition constraints (e.g., thepresence or absence of one of the four bases in the strand) can reducethis further (see below).

Medium resolution instrumentation, including but not exclusively curvedfield reflectron or delayed extraction time-of-flight MS instruments,can also result in improved DNA detection for sequencing or diagnostics.Either of these are capable of detecting a 9 Da (Δm (A-T)) shift in≧30-mer strands generated from, for example primer oligo base extension(PROBE), or competitive oligonucleotide single base extension (COSBE),sequencing, or direct detection of small amplified products.

Biomass Scan

In this embodiment, exemplified in Example 33, two single strandednucleic acids are individually immobilized to solid supports. Onesupport contains a nucleic acid encoding the wild type sequence whereasthe other support contains a nucleic acid encoding a mutant targetsequence. Total human genomic DNA is digested with one or morerestriction endonuclease enzyme resulting in the production of smallfragments of double stranded genomic DNA (10-1,000 bp). The digested DNAis incubated with the immobilized single stranded nucleic acids and thesample is heated to denature the DNA duplex. The immobilized nucleicacid competes with the other genomic DNA strand for the complementaryDNA strand and under the appropriate conditions, a portion of thecomplementary DNA strand hybridizes to the immobilized nucleic acidresulting in a strand displacement. By using high stringency washingconditions, the two nucleic acids will remain as a DNA duplex only ifthere is exact identity between the immobilized nucleic acid and thegenomic DNA strand. The DNA that remains hybridized to the immobilizednucleic acid is analyzed by mass spectrometry and detection of a signalin the mass spectrum of the appropriate mass is diagnostic for the wildtype or mutant allele. In this manner, total genomic DNA can be isolatedfrom a biological sample and screened for the presence or absence ofcertain mutations. By immobilizing a variety of single stranded nucleicacids in an array format, a panel of mutations may be simultaneouslyscreened for a number of genetic loci (i.e., multiplexing).

In addition, using less stringent washing conditions the hybridized DNAstrand may be analyzed by mass spectrometry for changes in the massresulting from a deletion or insertion within the targeted restrictionendonuclease fragment.

Primer Oligonucleotide Base Extension

As described in detail in the following Example 11, the primer oligobase extension (PROBE) method combined with mass spectrometry identifiesthe exact number of repeat units (i.e. the number of nucleotides inhomogenous stretches) as well as second site mutations within apolymorphic region, which are otherwise only detectable by sequencing.Thus, the PROBE technique increases the total number of detectablealleles at a distinct genomic site, leading to a higher polymorphisminformation content (PIC) and yielding a far more definitiveidentification in for instance statistics-based analyses in paternity orforensics applications.

The method is based on the extension of a detection primer that annealsadjacent to a variable nucleotide tandem repeat (VNTR) or a polymorphicmononucleotide stretch using a DNA polymerase in the presence of amixture of deoxyNTPs and those dideoxyNTPs that are not present in thedeoxy form. The resulting products are evaluated and resolved byMALDI-TOF mass spectrometry without further labeling of the DNA. In asimulated routine application with 28 unrelated individuals, the masserror of this procedure using external calibration was in the worst case0.38% (56-mer), which is comparable to approximately 0.1 base accuracy;routine standard mass deviations are in the range of 0.1% (0.03 bases).Such accuracy with conventional electrophoretic methods is notrealistic, underscoring the value of PROBE and mass spectrometry inforensic medicine and paternity testing.

The ultra-high resolution of Fourier Transform mass spectrometry makespossible the simultaneous measurement of all reactions of a Sanger orMaxam Gilbert sequencing experiment, since the sequence may be read frommass differences instead of base counting from 4 tubes.

Additionally, the mass differences between adjacent bases generated fromunilateral degradation in a stepwise manner by an exonuclease can beused to read the entire sequence of fragments generated. Whereas UV orfluorescent measurements will not discriminate mixtures of thenucleoside/nucleotide which are generated when the exonuclease enzymegets out of phase, this is no problem with mass spectrometry since theresolving power in differentiating between the molecular mass of dA, dT,dG and dC is more than significant. The mass of the adjacent bases(i.e., nucleotides) can be determined, for example, using Fast AtomicBombardment (FAB) or Electronspray Ionization (ESI) mass spectrometry.

New mutation screening over an entire amplified product can be achievedby searching for mass shifted fragments generated in an endonucleasedigestion as described in detail in the following Examples 4 and 12.

Partial sequence information obtained from tandem mass spectrometry(MS^(n)) can place composition constraints as described in the precedingparagraph. For the 25-mer above, generation of two fragment ions formedby collisionally activated dissociation (CAD) which differ by 313 Dadiscounts T₄C₁₂G₉, which contains no A nucleotides; confirming more thana single A eliminates AT₃C₄G₁₆ as a possible composition.

MS^(n) can also be used to determined full or partial sequences oflarger DNAs; this can be used to detect, locate, and identify newmutations in a given gene region. Enzymatic digest products whose massesare correct need not be further analyzed; those with mass shifts couldbe isolated in real time from the complex mixture in the massspectrometer and partially sequenced to locate the new mutation.

Table I describes the mutation/polymorphism detection tests that havebeen developed.

TABLE I Mutation/Polymorphism Detection Tests Clinical Association GeneMutation/Polymorphism Cystic Fibrosis CFTR 38 disease causing mutationsin 14 exons/introns Heart Disease (Cholesterol Apo E 112R, 112C, 158R,158C Metabolism) Apo A-IV 347S, 347T, 360H, 360Q Apo B-100 3500Q, 3500RThyroid Cancer RET proto- C634W, C634T, C634R oncogene C634S, C634FSickle Cell Anemia/ beta-globin Sickle cell anemia S and C Thalassemia45 thalassemia alleles HIV Susceptibility CKR-5 32 bp deletion BreastCancer BRCA-2 2 bp (AG) deletion in exon 2 Susceptibility ThrombosisFactor V R506Q Arteriosclerosis GpIIIa L33P E-selectin S128RHypertension ACE I/D polymorphism

Detection of Mutations

Diagnosis of Genetic Diseases

The mass spectrometric processes described above can be used, forexample, to diagnose any of the more than 3000 genetic diseasescurrently known (e.g., hemophilias, thalassemias, Duchenne MuscularDystrophy (DMD), Huntington's Disease (HD), Alzheimer's Disease andCystic Fibrosis (CF)) or to be identified.

The following Example 3 provides a mass spectrometric method fordetecting a mutation (ΔF508) of the cystic fibrosis transmembraneconductance regulator gene (CFTR), which differs by only three basepairs (900 daltons) from the wild type of CFTR gene. As describedfurther in Example 3, the detection is based on a single-tube,competitive oligonucleotide single base extension (COSBE) reaction usinga pair of primers with the 3′-terminal base complementary to either thenormal or mutant allele. Upon hybridization and addition of a polymeraseand the nucleoside triphosphate one base downstream, only those primersproperly annealed (i.e, no 3′-terminal mismatch) are extended; productsare resolved by molecular weight shifts as determined by matrix assistedlaser desorption ionization time-of-flight mass spectrometry. For thecystic fibrosis ΔF508 polymorphism, 28-mer ‘normal’ (N) and 30-mer‘mutant’ (M) primers generate 29- and 31-mers for N and M homozygotes,respectively, and both for heterozygotes. Since primer and productmolecular weights are relatively low (<10 kDa) and the mass differencebetween these are at least that of a single ˜300 Da nucleotide unit, lowresolution instrumentation is suitable for such measurements.

Thermosequence cycle sequencing, as further described in Example 11, isalso useful for detecting a genetic disease.

In addition to mutated genes, which result in genetic disease, certainbirth defects are the result of chromosomal abnormalities such asTrisomy 21 (Down's Syndrome), Trisomy 13 (Patau Syndrome), Trisomy 18(Edward's Syndrome), Monosomy X (Turner's Syndrome) and other sexchromosome aneuploidies such as Klienfelter's Syndrome (XXY). Here,“house-keeping” genes encoded by the chromosome in question are presentin different quantity and the different amount of an amplified fragmentcompared to the amount in a normal chromosomal configuration can bedetermined by mass spectrometry.

Further, there is growing evidence that certain DNA sequences maypredispose an individual to any of a number of diseases such asdiabetes, arteriosclerosis, obesity, various autoimmune diseases andcancer (e.g., colorectal, breast, ovarian, lung). Also, the detection of“DNA fingerprints”, e.g., polymorphisms, such as “mini- andmicro-satellite sequences”, are useful for determining identity orheredity (e.g., paternity or maternity).

The following Examples 4 and 12 provide mass spectrometer based methodsfor identifying any of the three different isoforms of humanapolipoprotein E, which are coded by the E2, E3 and E4 alleles. Forexample, the molecular weights of DNA fragments obtained afterrestriction with appropriate restriction endonucleases can be used todetect the presence of a mutation and/or a specific allele.

Depending on the biological sample, the diagnosis for a genetic disease,chromosomal aneuploidy or genetic predisposition can be preformed eitherpre- or post-natally.

Diagnosis of Cancer

Preferred mass spectrometer-based methods for providing an earlyindication of the existence of a tumor or a cancer are provide herein.For example, as described in Example 13, the telomeric repeatamplification protocol (TRAP) in conjunction with telomerase specificextension of a substrate primer and a subsequent amplification of thetelomerase specific extension products by an amplification step using asecond primer complementary to the repeat structure was used to obtainextension ladders, that were easily detected by MALDI-TOF massspectrometry as an indication of telomerase activity and therefortumorigenesis.

Alternatively, as described in Example 14, expression of a tumor orcancer associated gene (e.g., human tyrosine 5-hydroxylase) via RT-PCRand analysis of the amplified products by mass spectrometry can be usedto detect the tumor or cancer (e.g., biosynthesis of catecholamine viatyrosine 5-hydroxylase is a characteristic of neuroblastoma).

Further, a primer oligo base extension reaction and detection ofproducts by mass spectrometry provides a rapid means for detecting thepresence of oncogenes, such as the RET proto oncogene codon 634, whichis related to causing multiple endocrine neoplasia, type II (MEN II), asdescribed in Example 15.

Diagnosis of Infection

Viruses, bacteria, fungi and other infectious organisms contain distinctnucleic acid sequences, which are different from the sequences containedin the host cell. Detecting or quantitating nucleic acid sequences thatare specific to the infectious organism is important for diagnosing ormonitoring infection. Examples of disease causing viruses that infecthumans and animals and which may be detected by the disclosed processesinclude: Retroviridae (e.g., human immunodeficiency viruses, such asHIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, see, e.g.,Ratner et al. (1985) Nature 313: 227-284; Wain-Hobson et al. (1985) Cell40: 9-17); HIV-2 (see, Guyader et al. (1987) Nature 328: 662-669European Patent Publication No. 0 269 520; Chakrabarti et al. (1987)Nature 328: 543-547; and European Patent Application No. 0 655 501); andother isolates, such as HIV-LP (International PCT application No. WO94/00562 entitled “A Novel Human Immunodeficiency Virus”; Picornaviridae(e.g., polio viruses, hepatitis A virus, (see, e.g., Gust et al. (1983)Intervirology 20:1-7); entero viruses, human coxsackie viruses,rhinoviruses, echoviruses); Calciviridae (e.g., strains that causegastroenteritis); Togaviridae (e.g., equine encephalitis viruses,rubella viruses); Flaviridae (e.g., dengue viruses, encephalitisviruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses);Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses);Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaanviruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae(hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbivirusesand rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpessimplex virus (HSV) 1 and 2, varicella zoster virus, cytomegaovirus(CMV), herpes viruses'); Poxyiridae (variola viruses, vaccinia viruses,pox viruses); and Iridoviridae (e.g., African swine fever virus); andunclassified viruses (e.g., the etiological agents of Spongiformencephalopathies, the agent of delta hepatitis (thought to be adefective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses).

Examples of infectious bacteria include, but are not limited to:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcusphhenumoniae, pathogenic Campylobacter sp., Enterococcus sp.,Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, and Actinomyces israelli.

Examples of infectious fungi include: Cryptococcus neoformans,Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis,Chlamydia trachomatis, Candida albicans. Other infectious organisms(i.e., protists) include: Plasmodium falciparum and Toxoplasma gondii.

The processes provided herein makes use of the known sequenceinformation of the target sequence and known mutation sites. Althoughnew mutations can also be detected. For example, as shown in FIG. 8,transcription of a nucleic acid molecule obtained from a biologicalsample can be specifically digested using one or more nucleases and thefragments captured on a solid support carrying the correspondingcomplementary nucleic acid sequences. Detection of hybridization and themolecular weights of the captured target sequences provide informationon whether and where in a gene a mutation is present. Alternatively, DNAcan be cleaved by one or more specific endonucleases to form a mixtureof fragments. Comparison of the molecular weights between wildtype andmutant fragment mixtures results in mutation detection.

Sequencing by Generation of Specifically Terminated Fragments

In another embodiment, an accurate sequence determination of arelatively large target nucleic acid, can be obtained by generatingspecifically terminated fragments from the target nucleic acid,determining the mass of each fragment by mass spectrometry and orderingthe fragments to determine the sequence of the larger target nucleicacid. In a preferred embodiment, the specifically terminated fragmentsare partial or complete base-specifically terminated fragments.

One method for generating base specifically terminated fragmentsinvolves using a base-specific ribonuclease after e.g., a transcriptionreaction. Preferred base-specific ribonucleases are selected from among:T₁-ribonuclease (G-specific), U₂-ribonuclease (A-specific),PhyM-ribonuclease U specific and ribonuclease A (U/C specific). Otherefficient and base-specific ribonucleases can be identified using theassay described in Example 16. Preferably modified nucleotides areincluded in the transcription reaction with unmodified nucleotides. Mostpreferably, the modified nucleotides and unmodified nucleotides areadded to the transcription reaction at appropriate concentrations, sothat both moieties are incorporated at a preferential rate of about 1:1.Alternatively, two separate transcriptions of the target DNA sequenceone with the modified and one with the unmodified nucleotides can beperformed and the results compared. Preferred modified nucleotidesinclude: boron or bromine modified nucleotides (Porter et al. (1995)Biochemistry 34:11963-11969; Hasan et al. (1996) Nucl. Acids Res. 24:2150-2157; Li et al. (1995) Nucleic Acids Res. 23: 4495-4501),α-thio-modified nucleotides, as well as mass-modified nucleotides asdescribed above.

Another method for generating base specifically terminated fragmentsinvolves performing a combined amplification and base-specifictermination reaction. For example, a combined amplification andtermination reaction can be performed using at least two differentpolymerase enzymes, each having a different affinity for the chainterminating nucleotide, so that polymerization by an enzyme withrelatively low affinity for the chain terminating nucleotide leads toexponential amplification whereas an enzyme with relatively highaffinity for the chain terminating nucleotide terminates thepolymerization and yields sequencing products.

The combined amplification and sequencing can be based on anyamplification procedure that employs an enzyme with polynucleotidesynthetic ability (e.g., polymerase). One preferred process, based onthe polymerase chain reaction (PCR), includes the following threethermal steps: 1) denaturing a double stranded (ds) DNA molecule at anappropriate temperature and for an appropriate period of time to obtainthe two single stranded (ss) DNA molecules (the template: sense andantisense strand); 2) contacting the template with at least one primerthat hybridizes to at least one ss DNA template at an appropriatetemperature and for an appropriate period of time to obtain a primercontaining ss DNA template; 3) contacting the primer containing templateat an appropriate temperature and for an appropriate period of timewith: (i) a complete set of chain elongating nucleotides, (ii) at leastone chain terminating nucleotide, (iii) a first DNA polymerase, whichhas a relatively low affinity towards the chain terminating nucleotide;and (iv) a second DNA polymerase, which has a relatively high affinitytowards the chain terminating nucleotide.

Steps 1)-3) can be sequentially performed for an appropriate number oftimes (cycles) to obtain the desired amount of amplified sequencingladders. The quantity of the base specifically terminated fragmentdesired dictates how many cycles are performed. Although an increasednumber of cycles results in an increased level of amplification, it mayalso detract from the sensitivity of a subsequent detection. It istherefore generally undesirable to perform more than about 50 cycles,and is more preferable to perform less than about 40 cycles (e.g., about20-30 cycles).

Another preferred process for simultaneously amplifying and chainterminating a nucleic acid sequence is based on strand displacementamplification (SDA) (see, e.g., Walker et al. (1994) Nucl. Acids Res.22: 2670-77; European Patent Publication Number 0 684 315 entitled“Strand Displacement Amplification Using Thermophilic Enzymes”). Inessence, this process involves the following three steps, whichaltogether constitute a cycle: 1) denaturing a double stranded (ds) DNAmolecule containing the sequence to be amplified at an appropriatetemperature and for an appropriate period of time to obtain the twosingle stranded (ss) DNA molecules (the template: sense and antisensestrand); 2) contacting the template with at least one primer (P), thatcontains a recognition/cleavage site for a restriction endonuclease (RE)and that hybridizes to at least one ss DNA template at an appropriatetemperature and for an appropriate period of time to obtain a primercontaining ss DNA template; 3) contacting the primer containing templateat an appropriate temperature and for an appropriate period of time with(i) a complete set of chain elongating nucleotides; (ii) at least onechain terminating nucleotide; (iii) a first DNA polymerase, which has arelatively low affinity towards the chain terminating nucleotide; (iv) asecond DNA polymerase, which has a relatively high affinity towards thechain terminating nucleotide; and (v) an RE that nicks the primerrecognition/cleavage site.

Steps 1)-3) can be sequentially performed for an appropriate number oftimes (cycles) to obtain the desired amount of amplified sequencingladders. As with the PCR based process, the quantity of the basespecifically terminated fragment desired dictates how many cycles areperformed. Preferably, less than 50 cycles, more preferably less thanabout 40 cycles and most preferably about 20 to 30 cycles are performed.

Preferably about 0.5 to about 3 units of polymerase is used in thecombined amplification and chain termination reaction. Most preferablyabout 1 to 2 units is used. Particularly preferred polymerases for usein conjunction with PCR or other thermal amplification process arethermostable polymerases, such as Taq DNA polymerase (BoehringerMannheim), AmpliTaq FS DNA polymerase (Perkin-Elmer), Deep Vent (exo−),Vent, Vent (exo−) and Deep Vent DNA polymerases (New England Biolabs),Thermo Sequenase (Amersham) or exo(−) Pseudococcus furiosus (Pfu) DNApolymerase (Stratagene, Heidelberg, Germany). AmpliTaq, Ultman, 9 degreeNm, Tth, Hot Tub, and Pyrococcus furiosus. In addition, preferably thepolymerase does not have 5′-3′ exonuclease activity.

In addition to polymerases, which have a relatively high and arelatively low affinity to the chain terminating nucleotide, a thirdpolymerase, which has proofreading capacity (e.g., Pyrococcus woesei(Pwo)) DNA polymerase may also be added to the amplification mixture toenhance the fidelity of amplification.

Yet another method for generating base specifically terminated fragmentsinvolves contacting an appropriate amount of the target nucleic acidwith a specific endonuclease or exonuclease. Preferably, the original 5′and/or 3′ end of the nucleic acid is tagged to facilitate the orderingof fragments. Tagging of the 3′ end is particularly preferred when invitro nucleic acid transcripts are being analyzed, so that the influenceof 3′ heterogeneity, premature termination and nonspecific elongationcan be minimized. 5′ and 3′ tags can be natural (e.g., a 3′ poly A tailor 5′ or 3′ heterogeneity) or artificial. Preferred 5′ and/or 3′ tagsare selected from among the molecules described for mass-modificationabove.

The methods provided herein are further illustrated by the followingexamples, which should not be construed as limiting in any way.

EXAMPLE 1 MALDI-TOF Desorption of Oligonucleotides Directly on SolidSupports

1 g CPG (Controlled Pore Glass) was functionalized with3-(triethoxysilyl)-epoxypropan to form OH-groups on the polymer surface.A standard oligonucleotide synthesis with 13 mg of the OH-CPG on a DNAsynthesizer (Milligen, Model 7500) employingβ-cyanoethyl-phosphoamidites (Köster et al. (1994) Nucleic Acids Res.12: 4539) and TAC N-protecting groups (Köster et al. (1981) Tetrahedron37: 362) was performed to synthesize a 3′-T₅-50mer oligonucleotidesequence in which 50 nucleotides are complementary to a “hypothetical”50mer sequence. T₅ serves as a spacer. Deprotection with saturatedammonia in methanol at room temperature for 2 hours furnished accordingto the determination of the DMT group CPG which contained about 10 umol55mer/g CPG. This 55mer served as a template for hybridizations with a26-mer (with 5′-DMT group) and a 40-mer (without DMT group). Thereaction volume is 100 μl and contains about 1 nmol CPG bound 55mer astemplate, an equimolar amount of oligonucleotide in solution (26-mer or40-mer) in 20 mM Tris-HCl, pH 7.5, 10 mM MgCl₂ and 25 mM NaCl. Themixture was heated for 10 min at 65° C. and cooled to 37° C. during 30′(annealing). The oligonucleotide which has not been hybridized to thepolymer-bound template were removed by centrifugation and threesubsequent washing/centrifugation steps with 100 ul each of ice-cold 50mM ammoniumcitrate. The beads were air-dried and mixed with matrixsolution (3-hydroxypicolinic acid/10 mM ammonium citrate inacetonitrile/water, 1:1), and analyzed by MALDI-TOF mass spectrometry.The results are presented in FIGS. 10 and 11.

EXAMPLE 2 Electrospray (ES) Desorption and Differentiation of an 18-Merand 19-Mer

DNA fragments at a concentration of 50 pmole/ul in 2-propanol/10 mMammoniumcarbonate (1/9, v/v) were analyzed simultaneously by anelectrospray mass spectrometer.

The successful desorption and differentiation of an 18-mer and 19-mer byelectrospray mass spectrometry is shown in FIG. 12.

EXAMPLE 3 Detection of The Cystic Fibrosis Mutation ΔF508, by SingleStep Dideoxy Extension and Analysis by MALDI-TOF Mass SpectrometryCompetitive Oligonucleotide Simple Base Extension=COSBE

The principle of the COSBE method is shown in FIG. 13, N being thenormal and M the mutation detection primer, respectively.

Materials and Methods

PCR Amplification and Strand Immobilization. Amplification was carriedout with exon 10 specific primers using standard PCR conditions (30cycles: 1′@95° C., 1′@55° C., 2′@72° C.); the reverse primer was 5′labelled with biotin and column purified (Oligopurification Cartridge,Cruachem). After amplification the amplified products were purified bycolumn separation (Qiagen Quickspin) and immobilized on streptavidincoated magnetic beads (Dynabeads, Dynal, Norway) according to theirstandard protocol; DNA was denatured using 0.1 M NaOH and washed with0.1M NaOH, 1×B+W buffer and TE buffer to remove the non-biotinylatedsense strand.

COSBE Conditions. The beads containing ligated antisense strand wereresuspended in 18 μl of Reaction mix 1 (2 μl 10× Taq buffer, 1 μL (1unit) Taq Polymerase, 2 μL of 2 mM dGTP, and 13 μL H₂O) and incubated at80° C. for 5′ before the addition of Reaction mix 2 (100 ng each ofCOSBE primers). The temperature was reduced to 60° C. and the mixturesincubated for a 5′ annealing/extension period; the beads were thenwashed in 25 mM triethylammonium acetate (TEAA) followed by 50 mMammonium citrate.

Primer Sequences. All primers were synthesized on a PerspectiveBiosystems Expedite 8900 DNA Synthesizer using conventionalphosphoramidite chemistry (Sinha et al. (1984) Nucleic Acids Res. 12:4539). COSBE primers (each containing an intentional mismatch one basebefore the 3′-terminus) were those used in a previous ARMS study (Ferrieet al. (1992) Am J Hum Genet 51: 251-262) with the exception that twobases were removed from the 5′-end of the normal:

Ex10 PCR (Forward): (SEQ ID NO: 1) 5′-BIO-GCA AGT GAA TCC TGA GCG TG-3′Ex10 PCR (Reverse): (SEQ ID NO: 2) 5′-GTG TGA AGG GTT CAT ATG C-3′ COSBEΔF508-N (SEQ ID NO: 3) 5′-ATC TAT ATT CAT CAT AGG AAA CAC CAC A-3′(28-mer) COSBE ΔF508-N (SEQ ID NO: 4) 5′-GTA TCT ATA TTC ATC ATA GGA AACACC ATT-3′ (30-mer)

Mass Spectrometry. After washing, beads were resuspended in 1 μL 18Mohm/cm H₂O. 300 nL each of matrix (Wu et al. (1993) Rapid Commun. MassSpectrom. 7: 142-146) solution (0.7 M 3-hydroxypicolinic acid, 0.7 Mdibasic ammonium citrate in 1:1 H₂O:CH₃CN) and resuspended beads (Tanget al. (1995) Rapid Commun Mass Spectrom 8: 727-730) were mixed on asample target and allowed to air dry. Up to 20 samples were spotted on aprobe target disk for introduction into the source region of anunmodified Thermo Bioanalysis (formerly Finnigan) Visions 2000 MALDI-TOFoperated in reflectron mode with 5 and 20 kV on the target andconversion dynode, respectively. Theoretical average molecular weights(M_(r)(calc)) were calculated from atomic compositions. Vendor providedsoftware was used to determine peak centroids using externalcalibration; 1.08 Da has been subtracted from these to correct for thecharge carrying proton mass to yield the text M_(r)(exp) values.

Scheme. Upon annealing to the bound template, the N and M primers(8508.6 and 9148.0 Da, respectively) are presented with dGTP; onlyprimers with proper Watson-Crick base paring at the variable (V)position are extended by the polymerase. Thus if V pairs with the3′-terminal base of N, N is extended to a 8837.9 Da product (N+1).Likewise, if V is properly matched to the M terminus, M is extended to a9477.3 Da M+1 product.

Results

FIGS. 14-18 show the representative mass spectra of COSBE reactionproducts. Better results were obtained when amplified products werepurified before the biotinylated anti-sense strand was bound.

EXAMPLE 4 Differentiation of Human Apolipoprotein E Isoforms by MassSpectrometry

Apolipoprotein E (Apo E), a protein component of lipoproteins, plays anessential role in lipid metabolism. For example, it is involved withcholesterol transport, metabolism of lipoprotein particles,immunoregulation and activation of a number of lipolytic enzymes.

There are three common isoforms of human Apo E (coded by E2, E3 and E4alleles). The most common is the E3 allele. The E2 allele has been shownto decrease the cholesterol level in plasma and therefore may have aprotective effect against the development of atherosclerosis. The DNAencoding a portion of the E2 allele is set forth in SEQ ID NO: 130.Finally, the E4 isoform has been correlated with increased levels ofcholesterol, conferring predisposition to atherosclerosis. Therefore,the identity of the apo E allele of a particular individual is animportant determinant of risk for the development of cardiovasculardisease.

As shown in FIG. 19, a sample of DNA encoding apolipoprotein E can beobtained from a subject, amplified (e.g., via PCR); and the amplifiedproduct can be digested using an appropriate enzyme (e.g., CfoI). Therestriction digest obtained can then be analyzed by a variety of means.As shown in FIG. 20, the three isotypes of apolipoprotein E (E2, E3 andE4 have different nucleic acid sequences and therefore also havedistinguishable molecular weight values.

As shown in FIG. 21A-C, different Apolipoprotein E genotypes exhibitdifferent restriction patterns in a 3.5% MetPhor Agarose Gel or 12%polyacrylamide gel. As shown in FIGS. 22 and 23, the variousapolipoprotein E genotypes can also be accurately and rapidly determinedby mass spectrometry.

EXAMPLE 5 Detection of Hepatitis B Virus in Serum Samples Materials andMethods

Sample Preparation

Phenol/chloroform extraction of viral DNA and the final ethanolprecipitation was done according to standard protocols.

First PCR

Each reaction was performed with 5 μl of the DNA preparation from serum.15 pmol of each primer and 2 units Taq DNA polymerase (Perkin Elmer,Weiterstadt, Germany) were used. The final concentration of each dNTPwas 200 μMM, the final volume of the reaction was 50 μl. 10×PCR buffer(Perkin Elmer, Weiterstadt, Germany) contained 100 mM Tris-HCl, pH 8.3,500 mM KCl, 15 mM MgCl₂, 0.01% gelatine (w/v). Primer sequences:

SEQ ID Primer SEQUENCE NO: 1 5′-GCTTTGGGGCATGGACATTGACCCGTATAA-3′ 5 25′-CTGACTACTAATTCCCTGGATGCTGGGTCT-3′ 6

Nested PCR:

Each reaction was performed either with 1 μl of the first reaction orwith a 1:10 dilution of the first PCR as template, respectively. 100pmol of each primer, 2.5 u Pfu(exo−) DNA polymerase (Stratagene,Heidelberg, Germany), a final concentration of 200 μM of each dNTPs and5 μl 10×Pfu buffer (200 mM Tris-HCl, pH 8.75, 100 mM KCl, 100 mM(NH₄)₂SO₄, 1% Triton X-100, 1 mg/ml BSA, (Stratagene, Heidelberg,Germany) were used in a final volume 50 μl. The reactions were performedin a thermocycler (OmniGene, MWG-Biotech, Ebersberg, Germany) using thefollowing program: 92° C. for 1 minute, 60° C. for 1 minute and 72° C.for 1 minute with 20 cycles. Sequence of oligodeoxynucleotides(purchased HPLC-purified from MWG-Biotech, Ebersberg, Germany):

HBV13: 5′-TTGCCTGAGTGCAGTATGGT-3′ (SEQ ID NO: 7) HBV15bio:Biotin-5′-AGCTCTATATCGGGAAGCCT-3′ (SEQ ID NO: 8)

Purification of Amplified Products:

For the recording of each spectrum, one PCR, 50 μl, (performed asdescribed above) was used. Purification was done according to thefollowing procedure: Ultrafiltration was done using Ultrafree-MCfiltration units (Millipore, Eschborn, Germany) according to theprotocol of the provider with centrifugation at 8000 rpm for 20 minutes.25 μl (10 μg/μl) streptavidin Dynabeads (Dynal, Hamburg, Germany) wereprepared according to the instructions of the manufacturer andresuspended in 25 μl of B/W buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2M NaCl). This suspension was added to the PCR samples still in thefiltration unit and the mixture was incubated with gentle shaking for 15minutes at ambient temperature. The suspension was transferred in a 1.5ml Eppendorf tube and the supernatant was removed with the aid of aMagnetic Particle Collector, MPC, (Dynal, Hamburg, Germany). The beadswere washed twice with 50 μl of 0.7 M ammonium citrate solution, pH 8.0(the supernatant was removed each time using the MPC). Cleavage from thebeads can be accomplished by using formamide at 90° C. The supernatantwas dried in a speedvac for about an hour and resuspended in 4 μl ofultrapure water (MilliQ UF plus Millipore, Eschborn, Germany). Thispreparation was used for MALDI-TOF MS analysis.

MALDI-TOF MS:

Half a microliter of the sample was pipetted onto the sample holder,then immediately mixed with 0.5 μl matrix solution (0.7M3-hydroxypicolinic acid 50% acetonitrile, 70 mM ammonium citrate). Thismixture was dried at ambient temperature and introduced into the massspectrometer. All spectra were taken in positive ion mode using aFinnigan MAT Vision 2000 (Finnigan MAT, Bremen, Germany), equipped witha reflectron (5 keV ion source, 20 keV postacceleration) and a 337 nmnitrogen laser. Calibration was done with a mixture of a 40-mer and a100-mer. Each sample was measured with different laser energies. In thenegative samples, the amplified product was detected neither with lessnor with higher laser energies. In the positive samples the amplifiedproduct was detected at different places of the sample spot and alsowith varying laser energies.

Results

A nested PCR system was used for the detection of HBV DNA in bloodsamples employing oligonucleotides complementary to the c region of theHBV genome (primer 1: beginning at map position 1763, primer 2 beginningat map position 2032 of the complementary strand) encoding the HBV coreantigen (HBVcAg). DNA was isolated from patients serum according tostandard protocols. A first PCR was performed with the DNA from thesepreparations using a first set of primers. If HBV DNA was present in thesample a DNA fragment of 269 bp was generated.

In the second reaction, primers which were complementary to a regionwithin the PCR fragment generated in the first PCR were used. If HBVrelated amplified products were present in the first PCR a DNA fragmentof 67 bp was generated (see FIG. 25A) in this nested PCR. The usage of anested PCR system for detection provides a high sensitivity and alsoserves as a specificity control for the external PCR (Rolfs et al.(1992) PCR: Clinical Diagnostics and Research, Springer, Heidelberg). Afurther advantage is that the amount of fragments generated in thesecond PCR is high enough to ensure an unproblematic detection althoughpurification losses can not be avoided.

The samples were purified using ultrafiltration to restreptavidinDynabeads. This purification was done because the shorter primerfragments were immobilized in higher yield on the beads due to stearicreasons. The immobilization was done directly on the ultrafiltrationmembrane to avoid substance losses due to unspecific absorption on themembrane. Following immobilization, the beads were washed with ammoniumcitrate to perform cation exchange (Pieles et al. (1993) Nucl. AcidsRes. 21: 3191-3196). The immobilized DNA was cleaved from the beadsusing 25% ammonia which allows cleavage of DNA from the beads in a veryshort time, but does not result in an introduction of sodium or othercations.

The nested PCRs and the MALDI TOF analysis were performed withoutknowing the results of serological analysis. Due to the unknown virustiter, each sample of the first PCR was used undiluted as template andin a 1:10 dilution, respectively.

Sample 1 was collected from a patient with chronic active HBV infectionwho was positive in Hbs- and Hbe-antigen tests but negative in a dotblot analysis. Sample 2 was a serum sample from a patient with an activeHBV infection and a massive viremia who was HBV positive in a dot blotanalysis. Sample 3 was a denatured serum sample therefore no serologicalanalysis could be performed by an increased level of transaminasesindicating liver disease was detected. In autoradiograph analysis (FIG.24), the first PCR of this sample was negative. Nevertheless, there wassome evidence of HBV infection. This sample is of interest for MALDI-TOFanalysis, because it demonstrates that even low-level amounts ofamplified products can be detected after the purification procedure.Sample 4 was from a patient who was cured of HBV infection. Samples 5and 6 were collected from patients with a chronic active HBV infection.

FIG. 24 shows the results of a PAGE analysis of the nested PCR reaction.A amplified product is clearly revealed in samples 1, 2, 3, 5 and 6. Insample 4 no amplified product was generated, it is indeed HBV negative,according to the serological analysis. Negative and positive controlsare indicated by + and −, respectively. Amplification artifacts arevisible in lanes 2, 5, 6 and + if non-diluted template was used. Theseartifacts were not generated if the template was used in a 1:10dilution. In sample 3, amplified product was merely detectable if thetemplate was not diluted. The results of PAGE analysis are in agreementwith the data obtained by serological analysis except for sample 3 asdiscussed above.

FIG. 25A shows a mass spectrum of a nested amplified product from samplenumber 1 generated and purified as described above. The signal at 20754Da represents the single stranded amplified product (calculated: 20735Da, as the average mass of both strands of the amplified product cleavedfrom the beads). The mass difference of calculated and obtained mass is19 Da (0.09%). As shown in FIG. 25A, sample number 1 generated a highamount of amplified product, resulting in an unambiguous detection.

FIG. 25B shows a spectrum obtained from sample number 3. As depicted inFIG. 24, the amount of amplified product generated in this section issignificantly lower than that from sample number 1. Nevertheless, theamplified product is clearly revealed with a mass of 20751 Da(calculated 20735). The mass difference is 16 Da (0.08%). The spectrumdepicted in FIG. 25C was obtained from sample number 4 which is HBVnegative (as is also shown in FIG. 24). As expected no signalscorresponding to the amplified product could be detected. All samplesshown in FIG. 25 were analyzed with MALDI-TOF MS, whereby amplifiedproduct was detected in all HBV positive samples, but not in the HBVnegative samples. These results were reproduced in several independentexperiments.

EXAMPLE 6 Analysis of Ligase Chain Reaction Products Via MALDI-TOF MassSpectrometry Materials and Methods

Oligodeoxynucleotides

Except the biotinylated one and all other oligonucleotides weresynthesized in a 0.2 μmol scale on a MilliGen 7500 DNA Synthesizer(Millipore, Bedford, Mass., USA) using the β-cyanoethylphosphoamiditemethod (Sinha, N. D. et al. (1984) Nucleic Acids Res. 12: 4539-4577).The oligodeoxynucleotides were RP-HPLC-purified and deprotectedaccording to standard protocols. The biotinylated oligodeoxynucleotidewas purchased (HPLC-purified) from Biometra, Gottingen, Germany).Sequences and calculated masses of the oligonucleotides used:

SEQ Oligodeoxy- ID nucleotide SEQUENCE NO: A5′-p-TTGTGCCACGCGGTTGGGAATGTA 9 (7521 Da) B5′-p-AGCAACGACTGTTTGCCCGCCAGTTG 10 (7948 Da) C5′-bio-TACATTCCCAACCGCGTGGCACAAC 11 (7960 Da) D5′-p-AACTGGCGGGCAAACAGTCGTTGCT 12 (7708 Da)

5-Phosphorylation of Oligonucleotides A and D

This was performed with polynucleotide kinase (Boehringer, Mannheim,Germany) according to published procedures, the 5′-phosphorylatedoligonucleotides were used unpurified for LCR.

Ligase Chain Reaction

The LCR was performed with Pfu DNA ligase and a ligase chain reactionkit (Stratagene, Heidelberg, Germany) containing two differentpBluescript KII phagemids. One carrying the wildtype form of the E. colilacI gene and the other one a mutant of this gene with a single pointmutation at bp 191 of the lacI gene.

The following LCR conditions were used for each reaction: 100 pgtemplate DNA (0.74 fmol) with 500 pg sonified salmon sperm DNA ascarrier, 25 ng (3.3 pmol) of each 5′-phosphorylated oligonucleotide, 20ng (2.5 pmol) of each non-phosphorylated oligonucleotide, 4 U Pfu DNAligase in a final volume of 20 μl buffered ss 50-mer was used (I fmol)as template, in this case oligo C was also biotinylated. All reactionswere performed in a thermocycler (OmniGene, MWG-Biotech, Ebersberg,Germany) with the following program: 4 minutes 92° C., 2 minutes 60° C.and 25 cycles of 20 seconds 92° C., 40 seconds 60° C. Except for HPLCanalysis the biotinylated ligation educt C was used. In a controlexperiment the biotinylated and non-biotinylated oligonucleotidesrevealed the same gel electrophoretic results. The reactions wereanalyzed on 7.5% polyacrylamide gels. Ligation product 1 (oligo A and B)calculated mass: 15450 Da, ligation product 2 (oligo C and D) calculatedmass: 15387 Da.

SMART-HPLC

Ion exchange HPLC (IE HPLC) was performed on the SMART-system(Pharmacia, Freiburg, Germany) using a Pharmacia Mono Q, PC 1.6/5column. Eluents were buffer A (25 mM Tris-HCl, 1 mM EDTA and 0.3 M NaClat pH 8.0) and buffer B (same as A, but 1 M NaCl). Starting with 100% Afor 5 minutes at a flow rate of 50 μl/min. a gradient was applied from 0to 70% B in 30 minutes, then increased to 100% B in 2 minutes and heldat 100% B for 5 minutes. Two pooled LCR volumes (40 μl) performed witheither wildtype or mutant template were injected.

Sample Preparation for MALDI-TOF-MS

Preparation of Immobilized DNA: for the Recording of Each spectrum twoLCRs (performed as described above) were pooled and diluted 1:1 with2×B/W buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl). To thesamples 5 μl streptavidin DynaBeads (Dynal, Hamburg, Germany) wereadded, the mixture was allowed to bind with gentle shaking for 15minutes at ambient temperature. The supernatant was removed using aMagnetic Particle Collector, MPC, (Dynal, Hamburg, Germany) and thebeads were washed twice with 50 μl of 0.7 M ammonium citrate solution(pH 8.0) (the supernatant was removed each time using the MPC). Thebeads were resuspended in 1 μl of ultrapure water (MilliQ, Millipore,Bedford, Mabelow).

Combination of ultrafiltration and streptavidin DynaBeads: For therecording of spectrum two LCRs (performed as described above) werepooled, diluted 1:1 with 2×B/W buffer and concentrated with a 5000 NMWLUltrafree-MC filter unit (Millipore, Eschborn, Germany) according to theinstructions of the manufacturer. After concentration the samples werewashed with 300 μl 1×B/W buffer to streptavidin DynaBeads were added.The beads were washed once on the Ultrafree-MC filtration unit with 300μl of 1×B/W buffer and processed as described above. The beads wereresuspended in 30 to 50 μl of 1×B/W buffer and transferred in a 1.5 mlEppendorf tube. The supernatant was removed and the beads were washedtwice with 50 μl of 0.7 M ammonium citrate (pH 8.0). Finally, the beadswere washed once with 30 μl of acetone and resuspended in 1 μl ofultrapure water. The ligation mixture after immobilization on the beadswas used for MALDS-TOF-MS analysis as described below.

MALDI-TOF-MS

A suspension of streptavidin-coated magnetic beads with the immobilizedDNA was pipetted onto the sample holder, then immediately mixed with 0.5μl matrix solution (0.7 M 3-hydroxypicolinic acid in 50% acetonitrile,70 mM ammonium citrate). This mixture was dried at ambient temperatureand introduced into the mass spectrometer. All spectra were taken inpositive ion mode using a Finnigan MAT Vision 2000 (Finnigan MAT,Bremen, Germany), equipped with a reflectron (5 keV ion source, 20 keVpostacceleration) and a nitrogen laser (337 nm). For the analysis of PfuDNA ligase 0.5 μl of the solution was mixed on the sample holder with 1μl of matrix solution and prepared as described above. For the analysisof unpurified LCRs 1 μl of an LCR was mixed with 1 μl matrix solution.

Results

The E. coli lacI gene served as a simple model system to investigate thesuitability of MALDI-TOF-MS as detection method for products generatedin ligase chain reactions. This template system contains of an E. colilacI wildtype gene in a pBluescript KII phagemid and an E. coli lacIgene carrying a single point mutation at bp 191 (C to T transition; SEQID NO: 131) in the same phagemid. Four different oligonucleotides wereused, which were ligated only if the E coli lacI wildtype gene waspresent (FIG. 26).

LCR conditions were optimized using Pfu DNA ligase to obtain at least 1pmol ligation product in each positive reaction. The ligation reactionswere analyzed by polyacrylamide gel electrophoresis (PAGE) and HPLC onthe SMART system (FIGS. 27, 28 and 29). FIG. 27 shows a PAGE of apositive LCR with wildtype template (lane 1), a negative LCR with mutanttemplate (1 and 2) and a negative control which contains enzyme,oligonucleotides and no template but salmon sperm DNA. The gelelectrophoresis clearly shows that the ligation product (50 bp) wasproduced only in the reaction with wildtype template; whereas neitherthe template carrying the point mutation nor the control reaction withsalmon sperm DNA generated amplification products. In FIG. 28, HPLC wasused to analyze two pooled LCRs with wildtype template performed underthe same conditions. The ligation product was clearly revealed. FIG. 29shows the results of a HPLC in which two pooled negative LCRs withmutant template were analyzed. These chromatograms confirm the datashown in FIG. 27 and the results taken together clearly demonstrate,that the system generates ligation products in a significant amount onlyif the wildtype template is provided.

Appropriate control runs were performed to determine retention times ofthe different compounds involved in the L CR experiments. These includethe four oligonucleotides (A, B, C, and D), a synthetic ds 50-mer (withthe same sequence as the ligation product), the wildtype template DNA,sonicated salmon sperm DNA and the Pfu DNA ligase in ligation buffer.

In order to test which purification procedure should be used before aLCR reaction can be analyzed by MALDI-TOF-MS, aliquots of an unpurifiedLCR (FIG. 30A) and aliquots of the enzyme stock solution (FIG. 30B) wereanalyzed with MALDI-TOF-MS. It turned out that appropriate samplepreparation is absolutely necessary since all signals in the unpurifiedLCR correspond to signals obtained in the MALDI-TOF-MS analysis of thePfu DNA ligase. The calculated mass values of oligo A and the ligationproduct are 7521 Da and 15450 Da, respectively. The data in FIG. 30 showthat the enzyme solution leads to mass signals which do interfere withthe expected signals of the ligation educts and products and thereforemakes an unambiguous signal assignment impossible. Furthermore, thespectra showed signals of the detergent Tween20 being part of the enzymestorage buffer which influences the crystallization behavior of theanalyte/matrix mixture in an unfavorable way.

In one purification format streptavidin-coated magnetic beads were used.As was shown in a recent paper, the direct desorption of DNA immobilizedby Watson-Crick base pairing to a complementary DNA fragment covalentlybound to the beads is possible and the non-biotinylated strand will bedesorbed exclusively (Tang et al. (1995) Nucleic Acids Res. 23:3126-3131). This approach in using immobilized ds DNA ensures that onlythe non-biotinylated strand will be desorbed. If non-immobilized ds DNAis analyzed both strands are desorbed (Tang et al. (1994) Rapid Comm.Mass Spectrom. 7: 183-186) leading to broad signals depending on themass difference of the two single strands. Therefore, employing thissystem for LCR only the non-ligated oligonucleotide A, with a calculatedmass of 7521 Da, and the ligation product from oligo A and oligo B(calculated mass: 15450 Da) will be desorbed if oligo C is biotinylatedat the 5′-end and immobilized on steptavidin-coated beads. This resultsin a simple and unambiguous identification of the LCR educts andproducts.

FIG. 31A shows a MALDI-TOF mass spectrum obtained from two pooled LCRs(performed as described above) purified on streptavidin DynaBeads anddesorbed directly from the beads showed that the purification methodused was efficient (compared with FIG. 30). A signal which representsthe unligated oligo A and a signal which corresponds to the ligationproduct could be detected. The agreement between the calculated and theexperimentally found mass values is remarkable and allows an unambiguouspeak assignment and accurate detection of the ligation product. Incontrast, no ligation product but only oligo A could be detected in thespectrum obtained from two pooled LCRs with mutated template (FIG. 31B).The specificity and selectivity of the LCR conditions and thesensitivity of the MALDI-TOF detection is further demonstrated whenperforming the ligation reaction in the absence of a specific template.FIG. 32 shows a spectrum obtained from two pooled LCRs in which onlysalmon sperm DNA was used as a negative control, only oligo A could bedetected, as expected.

While the results shown in FIG. 31A can be correlated to lane 1 of thegel in FIG. 27, the spectrum shown in FIG. 31B is equivalent to lane 2in FIG. 27, and finally also the spectrum in FIG. 32 corresponds to lane3 in FIG. 27. The results are in congruence with the HPLC analysispresented in FIGS. 28 and 29. While gel electrophoresis (FIG. 27) andHPLC (FIGS. 28 and 29) reveal either an excess or almost equal amountsof ligation product over ligation educts, the analysis by MALDI-TOF massspectrometry produces a smaller signal for the ligation product (FIG.31A).

The lower intensity of the ligation product signal could be due todifferent desorption/ionization efficiencies between 24- and a 50-mer.Since the T_(m) value of a duplex with 50 compared to 24 base pairs issignificantly higher, more 24-mer could be desorbed. A reduction insignal intensity can also result from a higher degree of fragmentationin case of the longer oligonucleotides.

Regardless of the purification with streptavidin DynaBeads, FIG. 32reveals traces of Tween20 in the region around 2000 Da. Substances witha viscous consistence, negatively influence the process ofcrystallization and therefore can be detrimental to mass spectrometeranalysis. Tween20 and also glycerol which are part of enzyme storagebuffers therefore should be removed entirely prior to mass spectrometeranalysis. For this reason an improved purification procedure whichincludes an additional ultrafiltration step prior to treatment withDynaBeads was investigated. Indeed, this sample purification resulted ina significant improvement of MALDI-TOF mass spectrometric performance.

FIG. 33 shows spectra obtained from two pooled positive (FIG. 33A) andnegative (FIG. 33B) LCRs, respectively. The positive reaction wasperformed with a chemically synthesized, single strand 50mer as templatewith a sequence equivalent to the ligation product of oligo C and D.Oligo C was 5′-biotinylated. Therefore the template was not detected. Asexpected, only the ligation product of Oligo A and B (calculated mass15450 Da) could be desorbed from the immobilized and ligated oligo C andD. This newly generated DNA fragment is represented by the mass signalof 15448 Da in FIG. 33A. Compared to FIG. 32A, this spectrum clearlyshows that this method of sample preparation produces signals withimproved resolution and intensity.

EXAMPLE 7 Mutation Detection by Solid Phase Oligo Base Extension of aPrimer and Analysis by MALDI-TOF Mass Spectrometry Primer Oligo BaseExtension=Probe

Summary

The solid-phase oligo base extension method detects point mutations andsmall deletions as well as small insertions in amplified DNA. The methodis based on the extension of a detection primer that anneals adjacent toa variable nucleotide position on an affinity-captured amplifiedtemplate, using a DNA polymerase, a mixture of three dNTPs, and themissing one dideoxy nucleotide. The resulting products are evaluated andresolved by MALDI-TOF mass spectrometry without further labelingprocedures. The aim of the following experiment was to determine mutantand wildtype alleles in a fast and reliable manner.

Description of the Experiment

The method used a single detection primer followed by a oligonucleotideextension step to give products differing in length by some basesspecific for mutant or wildtype alleles which can be easily resolved byMALDI-TOF mass spectrometry. The method is described by using as examplethe exon 10 of the CFTR-gene. Exon 10 of this gene bears the most commonmutation in many ethnic groups (ΔF508) that leads in the homozygousstate to the clinical phenotype of cystic fibrosis.

Materials and Methods

Genomic DNA

Genomic DNA were obtained from healthy individuals, individualshomozygous or heterozygous for the ΔF508 mutation, and one individualheterozygous for the 1506S mutation. The wildtype and mutant alleleswere confirmed by standard Sanger sequencing.

PCR Amplification of Exon 10 of the CFTR Gene

The primers for PCR amplification were CFEx10-F(5-GCAAGTGAATCCTGAGCGTG-3′ (SEQ ID NO: 13) located in intron 9 andbiotinylated) and CFEx10-R (5′-GTGTGAAGGGCGTG-3′ SEQ ID NO: 14) locatedin intron 10). Primers were used in a concentration of 8 pmol.Taq-polymerase including 10× buffer were purchased fromBoehringer-Mannheim and dTNPs were obtained from Pharmacia. The totalreaction volume was 50 μl. Cycling conditions for PCR were initially 5min. at 95° C., followed by 1 min. at 94° C., 45 sec at 53° C., and 30sec at 72° C. for 40 cycles with a final extension time of 5 min at 72°C.

Purification of the Amplified Products

Amplification products were purified by using Qiagen's PCR purificationkit (No. 28106) according to manufacturer's instructions. The elution ofthe purified products from the column was done in 50 μl TE-buffer (10 mMTris, 1 mM EDTA, pH 7.5).

Affinity-Capture and Denaturation of the Double Stranded DNA

10 μL aliquots of the purified amplified product were transferred to onewell of a streptavidin-coated microtiter plate (No. 1645684Boehringer-Mannheim or No. 95029262 Labsystems). Subsequently, 10 μlincubation buffer (80 mM sodium phosphate, 400 mM NaCl, 0.4% Tween20, pH7.5) and 30 μl water were added. After incubation for 1 hour at roomtemperature the wells were washed three times with 200 μl washing buffer(40 mM Tris, 1 mM EDTA, 50 mM NaCl, 0.1% Tween 20, pH 8.8). To denaturethe double stranded DNA the wells were treated with 100 μl of a 50 mMNaOH solution for 3 min and the wells washed three times with 200 μlwashing buffer.

Oligo Base Extension Reaction

The annealing of 25 pmol detection primer (CF508:5′-CTATATTCATCATAGGAAACACCA-3′ (SEQ ID NO: 15) was performed in 50 μlannealing buffer (20 mM Tris, 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO2, 1%Triton X-100, pH 8) at 50° C. for 10 min. The wells were washed threetimes with 200 μl washing buffer and once in 200 μl TE buffer. Theextension reaction was performed by using some components of the DNAsequencing kit from USB (No. 70770) and dNTPs or ddNTPs from Pharmacia.The total reaction volume was 45 μl, containing of 21 μl water, 6 μlSequenase-buffer, 3 μl 10 mM DTT solution, 4.5 μl, 0.5 mM of threedNTPs, 4.5 μl, 2 mM the missing one ddNTP, 5.5 μl glycerol enzymedilution buffer, 0.25 μl Sequenase 2.0, and 0.25 pyrophosphatase. Thereaction was pipetted on ice and then incubated for 15 min at roomtemperature and for 5 min at 37° C. Hence, the wells were washed threetimes with 200 μl washing buffer and once with 60 μl of a 70 mMNH₄-Citrate solution.

Denaturation and Precipitation of the Extended Primer

The extended primer was denatured in 50 μl 10%-DMSO (dimethylsulfoxide)in water at 80° C. for 10 min. For precipitation, 10 μl NH₄-Acetate (pH6.5), 0.5 μl glycogen (10 mg/ml water, Sigma No. G1765), and 100 μlabsolute ethanol were added to the supernatant and incubated for 1 hourat room temperature. After centrifugation at 13.000 g for 10 min thepellet was washed in 70% ethanol and resuspended in 1 μl 18 Mohm/cm H₂Owater.

Sample Preparation and Analysis on MALDI-TOF Mass Spectrometry

Sample preparation was performed by mixing 0.3 μl of each of matrixsolution (0.7 M 3-hydroxypicolinic acid, 0.07 M dibasic ammonium citratein 1:1 H₂O:CH₃CN) and of resuspended DNA/glycogen pellet on a sampletarget and allowed to air dry. Up to 20 samples were spotted on a probetarget disk for introduction into the source region of an unmodifiedThermo Bioanalysis (formerly Finnigan) Visions 2000 MALDI-TOF operatedin reflectron mode with 5 and 20 kV on the target and conversion dynode,respectively. Theoretical average molecular mass (M_(r)(calc)) werecalculated from atomic compositions; reported experimental Mr(M_(r)(exp)) values are those of the singly-protonated form, determinedusing external calibration.

Results

The aim of the experiment was to develop a fast and reliable methodindependent of exact stringencies for mutation detection that leads tohigh quality and high throughput in the diagnosis of genetic diseases.Therefore a special kind of DNA sequencing (oligo base extension of onemutation detection primer) was combined with the evaluation of theresulting mini-sequencing products by matrix-assisted laser desorptionionization (MALDI) mass spectrometry (MS). The time-of-flight (TOF)reflectron arrangement was chosen as a possible mass measurement system.To prove this hypothesis, the examination was performed with exon 10 ofthe CFTR-gene, in which some mutations could lead to the clinicalphenotype of cystic fibrosis, the most common monogenetic disease in theCaucasian population.

The schematic presentation as given in FIG. 34 shows the expected shortsequencing products with the theoretically calculated molecular mass ofthe wildtype and various mutations of exon 10 of the CFTR-gene (SEQ IDNO: 132). The short sequencing products were produced using either ddTTP(FIG. 34A; SEQ ID NOs: 133-135) or ddCTP (FIG. 34B; SEQ ID NOs: 136-139)to introduce a definitive sequence related stop in the nascent DNAstrand. The MALDI-TOF-MS spectra of healthy, mutation heterozygous, andmutation homozygous individuals are presented in FIG. 35. All sampleswere confirmed by standard Sanger sequencing which showed no discrepancyin comparison to the mass spec analysis. The accuracy of theexperimental measurements of the various molecular masses was within arange of minus 21.8 and plus 87.1 dalton (Da) to the range expected.This allows a definitive interpretation of the results in each case. Afurther advantage of this procedure is the unambiguous detection of theΔI507 mutation. In the ddTTP reaction, the wildtype allele would bedetected, whereas in the ddCTP reaction the three base pair deletionwould be disclosed.

The method described is highly suitable for the detection of singlepoint mutations or microlesions of DNA. Careful choice of the mutationdetection primers will open the window of multiplexing and lead to ahigh throughput including high quality in genetic diagnosis without anyneed for exact stringencies necessary in comparable allele-specificprocedures. Because of the uniqueness of the genetic information, theoligo base extension of mutation detection primer is applicable in eachdisease gene or polymorphic region in the genome like variable number oftandem repeats (VNTR) or other single nucleotide polymorphisms (e.g.,apolipoprotein E gene), as also described here.

EXAMPLE 8 Detection of Polymerase Chain Reaction Products Containing7-Deazapurine Moieties with Matrix-Assisted Laser Desorption/IonizationTime-of-Flight (MALDI-TOF) Mass Spectrometry Materials and Methods

Nucleic Acid Amplifications

The following oligodeoxynucleotide primers were either synthesizedaccording to standard phosphoamidite chemistry (Sinha, N. D., et al.,(1983) Tetrahedron Let. Vol. 24, Pp. 5843-5846; Sinha, N. D., et al.,(1984) Nucleic Acids Res., Vol. 12, Pp. 4539-4557) on a MilliGen 7500DNA synthesizer (Millipore, Bedford, Mass., USA) in 200 nmol scales orpurchased from MWG-Biotech (Ebersberg, Germany, primer 3) and Biometra(Goettingen, Germany, primers 6-7).

(SEQ ID NO: 16) primer 1: 5′-GTCACCCTCGACCTGCAG; (SEQ ID NO: 17) primer2: 5′-TTGTAAAACGACGGCCAGT; (SEQ ID NO: 18) primer 3:5′-CTTCCACCGCGATGTTGA; (SEQ ID NO: 19) primer 4: 5′-CAGGAAACAGCTATGAC;(SEQ ID NO: 20) primer 5: 5′-GTAAAACGACGGCCAGT; (SEQ ID NO: 21) primer6: 5′-GTCACCCTCGACCTGCAgG (g: RiboG); (SEQ ID NO: 22) primer 7:5′-GTTGTAAAACGAGGGCCAgT (g: RiboG);

The 99-mer (SEQ ID NO: 141) and 200-mer DNA strands (SEQ ID NO: 140;modified and unmodified) as well as the ribo- and 7-deaza-modified100-mer were amplified from pRFc1 DNA (10 ng, generously supplied by S.Feyerabend, University of Hamburg) in 100 μL reaction volume containing10 mmol/L KCl, 10 mmol/L (NH₄)₂SO₄, 20 mmol/L Tris HCl (pH 8.8), 2mmol/L MgSO₄₁ (exo(−) Pseudococcus furiosus (Pfu)-Buffer, Pharmacia,Freiburg, Germany), 0.2 mmol/L each dNTP (Pharmacia, Freiburg, Germany),1 μmol/L of each primer and 1 unit of exo(−)Pfu DNA polymerase(Stratagene, Heidelberg, Germany). For the 99-mer primers 1 and 2, forthe 200-mer primers 1 and 3 and for the 100-mer primers 6 and 7 wereused. To obtain 7-deazapurine modified nucleic acids, duringPCR-amplification dATP and dGTP were replaced with 7-deaza-dATP and7-deaza-dGTP. The reaction was performed in a thermal cycler (OmniGene,MWG-Biotech, Ebersberg, Germany) using the cycle: denaturation at 95° C.for 1 min., annealing at 51° C. for 1 min. and extension at 72° C. for 1min. For all PCRs the number of reaction cycles was 30. The reaction wasallowed to extend for additional 10 min. at 72° C. after the last cycle.

The 103-mer DNA strands (modified and unmodified; SEQ ID NO: 245) wereamplified from M13mp18 RFI DNA (100 ng, Pharmacia, Freiburg, Germany) in100 μL reaction volume. using primers 4 and 5 all other concentrationswere unchanged. The reaction was performed using the cycle: denaturationat 95° C. for 1 min., annealing at 40° C. for 1 min. and extension at72° C. for 1 min. After 30 cycles for the unmodified and 40 cycles forthe modified 103-mer respectively, the samples were incubated foradditional 10 min. at 72° C.

Synthesis of 5′-[³²-P]-Labeled PCR-Primers

Primers 1 and 4 were 5′-[³²-P]-labeled employing T4-polynucleotidekinase(Epicentre Technologies) and (γ-³²P)-ATP. (BLU/NGG/502A, Dupont,Germany) according to the protocols of the manufacturer. The reactionswere performed substituting 10% of primer 1 and 4 in PCR with thelabeled primers under otherwise unchanged reaction-conditions. Theamplified DNAs were separated by gel electrophoresis on a 10%polyacrylamide gel. The appropriate bands were excised and counted on aPackard TRI-CARB 460C liquid scintillation system (Packard, Conn., USA).

Primer-Cleavage from Ribo-Modified PCR-Product

The amplified DNA was purified using Ultrafree-MC filter units (30,000NMWL), it was then redissolved in 100 μl of 0.2 mol/L NaOH and heated at95° C. for 25 minutes. The solution was then acidified with HCl (1mol/L) and further purified for MALDI-TOF analysis employingUltrafree-MC filter units (10,000 NMWL) as described below.

Purification of Amplified Products

All samples were purified and concentrated using Ultrafree-MC units30000 NMWL (Millipore, Eschborn, Germany) according to themanufacturer's description. After lyophilization, amplified productswere redissolved in 5 μL (3 μL for the 200-mer) of ultrapure water. Thisanalyte solution was directly used for MALDI-TOF measurements.

MALDI-TOF MS

Aliquots of 0.5 μL of analyte solution and 0.5 μL of matrix solution(0.7 mol/L 3-HPA and 0.07 mol/L ammonium citrate in acetonitrile/water(1:1, v/v)) were mixed on a flat metallic sample support. After dryingat ambient temperature the sample was introduced into the massspectrometer for analysis. The MALDI-TOF mass spectrometer used was aFinnigan MAT Vision 2000 (Finnigan MAT, Bremen, Germany). Spectra wererecorded in the positive ion reflector mode with a 5 keV ion source and20 keV postacceleration. The instrument was equipped with a nitrogenlaser (337 nm wavelength). The vacuum of the system was 3-4·10⁸ hPa inthe analyzer region and 1-4·10⁻⁷ hPa in the source region. Spectra ofmodified and unmodified DNA samples were obtained with the same relativelaser power; external calibration was performed with a mixture ofsynthetic oligodeoxynucleotides (7- to 5O-mer).

Results and Discussion Enzymatic Synthesis of 7-Deazapurine NucleotideContaining Nucleic Acids by PCR

In order to demonstrate the feasibility of MALDI-TOF MS for the rapid,gel-free analysis of short amplified products and to investigate theeffect of 7-deazapurine modification of nucleic acids under MALDI-TOFconditions, two different primer-template systems were used tosynthesize DNA fragments. Sequences are displayed in FIGS. 36 and 37.While the two single strands of the 103-mer amplified product had nearlyequal masses (Δm=8 u), the two single strands of the 99-mer differed by526 u. Considering that 7-deaza purine nucleotide building blocks forchemical DNA synthesis are approximately 160 times more expensive thanregular ones (Product Information, Glen Research Corporation, Sterling,Va.) and their applification in standard β-cyano-phosphoamiditechemistry is not trivial (Product Information, Glen ResearchCorporation, Sterling, Va.; Schneider et al. (1995) Nuc. Acids Res. 23:1570) the cost of 7-deaza purine modified primers would be very high.Therefore, to increase the applicability and scope of the method, allPCRs were performed using unmodified oligonucleotide primers which areroutinely available. Substituting dATP and dGTP by c⁷-dATP and c⁷-dGTPin polymerase chain reaction led to products containing approximately80% 7-deaza-purine modified nucleosides for the 99-mer and 103-mer; andabout 90% for the 200-mer, respectively. Table II shows the basecomposition of all PCR products.

TABLE II Base composition of the 99-mer, 103-mer and 200-mer PCRamplification products (unmodified and 7-deaza purine modified)c⁷-deaza- rel. DNA-fragments¹ C T A G A c⁷-deaza-6 mod.2 200-mers 54 3456 56 — — — modified 200-mer s 54 34 6 5 50 51 90% 200-mer a 56 56 34 54— — — modified 200-mer a 56 56 3 4 31 50 92% 103-mer s 28 23 24 28 — — —modified 103-mer s 28 23 6 5 18 23 79% 103-mer a 28 24 23 28 — — —modified 103-mer a 28 24 7 4 16 24 78% 99-mer s 34 21 24 20 — — —modified 99-mer s 34 21 6 5 18 15 75% 99-mer a 20 24 21 34 — — —modified 99-mer a 20 24 3 4 18 30 87% ¹“s” and “a” describe “sense” and“antisense” strands of the double-stranded amplified product. ²indicatesrelative modification as percentage of 7-deaza purine modifiednucleotides of total amount of purine nucleotides.

It remained to be determined whether 80-90% 7-deaza-purine modificationis sufficient for accurate mass spectrometer detection. It was thereforeimportant to determine whether all purine nucleotides could besubstituted during the enzymatic amplification step. This was nottrivial since it had been shown that c⁷-dATP cannot fully replace dATPin PCR if Taq DNA polymerase is employed (Seela, F. and A. Roelling(1992) Nucleic Acids Res., 20, 55-61). Fortunately it was found thatexo(−)Pfu DNA polymerase indeed could accept c⁷-dATP and c⁷-dGTP in theabsence of unmodified purine nucleoside triphosphates. The incorporationwas less efficient leading to a lower yield of amplified product (FIG.38).

To verify these results, the amplifications with [³²P]-labeled primerswere repeated. The autoradiogram (FIG. 39) clearly shows lower yieldsfor the modified PCR-products. The bands were excised from the gel andcounted. For all amplified products the yield of the modified nucleicacids was about 50%, referring to the corresponding unmodifiedamplification product. Further experiments showed that exo(−) DeepVentand Vent DNA polymerase were able to incorporate c⁷-dATP and c⁷-dGTPduring PCR as well. The overall performance, however, turned out to bebest for the exo(−)Pfu DNA polymerase giving least side products duringamplification. Using all three polymerases, it was found that such PCRsemploying c⁷-dATP and c⁷-dGTP instead of their isosteres showed lessside-reactions giving a cleaner PCR-product. Decreased occurrence ofamplification side products may be explained by a reduction of primermismatches due to a ling template which is synthesized during PCR.Decreased melting point for DNA duplexes containing 7-deaza-purine havebeen described (Mizusawa, S. et al., (1986) Nucleic Acids Res., 14,1319-1324). In addition to the three polymerases specified above (exo(−)Deep Vent DNA polymerase, 5Vent DNA polymerase and exo(−) (Pfu) DNApolymerase), it is anticipated that other polymerases, such as the LargeKlenow fragment of E. coli DNA polymerase, Sequenase, Taq DNA polymeraseand U AmpliTaq DNA polymerase can be used. In addition, where RNA is thetemplate, RNA polymerases, such as the SP6 or the T7 RNA polymerase,must be used.

MALDI-TOF Mass Spectrometry of Modified and Unmodified AmplifiedProducts.

The 99-mer, 103-mer and 200-mer amplified products were analyzed byMALDI-TOF MS. Based on past experience, it was known that the degree ofdepurination depends on the laser energy used for desorption andionization of the analyte. Since the influence of 7-deazapurinemodification on fragmentation due to depurination was to beinvestigated, all spectra were measured at the same relative laserenergy.

FIGS. 40 a and 40 b show the mass spectra of the modified and unmodified103-mer nucleic acids. In case of the modified 103-mer, fragmentationcauses a broad (M+H)⁺ signal. The maximum of the peak is shifted tolower masses so that the assigned mass represents a mean value of (M+H)⁺signal and signals of fragmented ions, rather than the (M+H)⁺ signalitself. Although the modified 103-mer still contains about 20% A and Gfrom the oligonucleotide primers, it shows less fragmentation which isfeatured by much more narrow and symmetric signals. Especially peaktailing on the lower mass side due to depurination, is substantiallyreduced. Hence, the difference between measured and calculated mass isstrongly reduced although it is still below the expected mass. For theunmodified sample a (M+H)⁺ signal of 31670 was observed, which is a 97 uor 0.3% difference to the calculated mass. While, in case of themodified sample this mass difference diminished to 10 u or 0.03% (31713u found, 31723 u calculated). These observations are verified by asignificant increase in mass resolution of the (M+H)⁺ signal of the twosignal strands (n/Δm=67 as opposed to 18 for the unmodified sample withΔm=full width at half maximum, fwhm). Because of the low mass differencebetween the two single strands (8 u) their individual signals were notresolved.

With the results of the 99 base pair DNA fragments the effects ofincreased mass resolution for 7-deazapurine containing DNA becomes evenmore evident. The two single strands in the unmodified sample were notresolved even though the mass difference between the two strands of theamplified product was very high with 526 u due to unequal distributionof purines and pyrimidines (FIG. 41 a). In contrast to this, themodified DNA showed distinct peaks for the two single strands (FIG. 41b) which demonstrates the superiority of this approach for thedetermination of molecular weights to gel electrophoretic methods evenmore profound. Although base line resolution was not obtained theindividual masses were able to be assigned with an accuracy of 0.1%:Δm=27 u for the lighter (calc. mass=30224 u) and Δm=14 u for the heavierstrand (calc. mass=30750 u). Again, it was found that the full width athalf maximum was substantially decreased for the 7-deazapurinecontaining sample.

In case the 99-mer and 103-mer, the 7-deazapurine containing nucleicacids seem to give higher sensitivity despite the fact that they stillcontain about 20% unmodified purine nucleotides. To get comparablesignal-to-noise ratio at similar intensities for the (M+H)⁺ signals, theunmodified 99-mer required 20 laser shots in contrast to 12 for themodified one and the 103-mer required 12 shots for the unmodified sampleas opposed to three for the 7-deazapurine nucleoside-containingamplified product.

Comparing the spectra of the modified and unmodified 200-mer amplicons,improved mass resolution was again found for the 7-deazapurinecontaining sample as well as increased signal intensities (FIGS. 42A and42B). While the signal of the single strands predominates in thespectrum of the modified sample the DNA-duplex and dimers of the singlestrands gave the strongest signal for the unmodified sample.

A complete 7-deaza purine modification of nucleic acids may be achievedeither using modified primers in PCR or cleaving the unmodified primersfrom the partially modified amplified product. Since disadvantages areassociated with modified primers, as described above, a 100-mer wassynthesized using primers with a ribo-modification. The primers werecleaved hydrolytically with NaOH according to a method developed earlierin our laboratory (Koester, H. et al., Z Physiol. Chem., 359,1570-1589). FIGS. 43A and 43B display the spectra of the amplifiedproduct before and after primer cleavage. FIG. 43 b shows that thehydrolysis was successful: The hydrolyzed amplified product as well asthe two released primers could be detected together with a small signalfrom residual uncleaved 100-mer. This procedure is especially useful forthe MALDI-TOF analysis of very short PCR-products since the share ofunmodified purines originating from the primer increases with decreasinglength of the amplified sequence.

The remarkable properties of 7-deazapurine modified nucleic acids can beexplained by either more effective desorption and/or ionization,increased ion stability and/or a lower denaturation energy of the doublestranded purine modified nucleic acid. The exchange of the N-7 for amethyl group results in the loss of one acceptor for a hydrogen bondwhich influences the ability of the nucleic acid to form secondarystructures due to non-Watson-Crick base pairing (Seela, F. and A. Kehne(1987) Biochemistry, 26, 2232-2238). In addition to this the aromaticsystem of 7-deazapurine has a lower electron density that weakensWatson-Crick base pairing resulting in a decreased melting point(Mizusawa, S. et al., (1986) Nucleic Acids Res., 14, 1319-1324) of thedouble-strand. This effect may decrease the energy needed fordenaturation of the duplex in the MALDI process. These aspects as wellas the loss of a site which probably will carry a positive charge on theN-7 nitrogen renders the 7-deazapurine modified nucleic acid less polarand may promote the effectiveness of desorption.

Because of the absence of N-7 as proton acceptor and the decreasedpolarization of the C—N bond in 7-deazapurine nucleosides depurinationfollowing the mechanisms established for hydrolysis in solution isprevented. Although a direct correlation of reactions in solution and inthe gas phase is problematic, less fragmentation due to depurination ofthe modified nucleic acids can be expected in the MALDI process.Depurination may either be accompanied by loss of charge which decreasesthe total yield of charged species or it may produce chargedfragmentation products which decreases the intensity of the nonfragmented molecular ion signal.

The observation of increased sensitivity and decreased peak tailing ofthe (M+H)⁺ signals on the lower mass side due to decreased fragmentationof the 7-deazapurine containing samples indicate that the N-7 atomindeed is essential for the mechanism of depurination in the MALDI-TOFprocess. In conclusion, 7-deazapurine containing nucleic acids showdistinctly increased ion-stability and sensitivity under MALDI-TOFconditions and therefore provide for higher mass accuracy and massresolution.

EXAMPLE 9 Solid Phase Sequencing and Mass Spectrometer DetectionMaterials and Methods

Oligonucleotides were purchased from Operon Technologies (Alameda,Calif.) in an unpurified form. Sequencing reactions were performed on asolid surface using reagents from the sequencing kit for SequenaseVersion 2.0 (Amersham, Arlington Heights, Ill.).

Sequencing a 39-Mer Target

Sequencing complex:

SEQ ID SEQUENCE NO:5′-TCTGGCCTGGTGCAGGGCCTATTGTAGTTGTGACGTACA-(A^(b))_(a)-3′ 235′-TGTACGTCACAACT-3′ (PNA 16/DNA) 24

In order to perform solid-phase DNA sequencing, template strand DNA11683was 3′-biotinylated by terminal deoxynucleotidyl transferase. A 30 μlreaction, containing 60 pmol of DNA11683, 1.3 nmol of biotin 14-dATP(GIBCO BRL, Grand Island, N. Y.), 30 units of terminal transferase(Amersham, Arlington Heights, Ill.), and 1× reaction buffer (suppliedwith enzyme), was incubated at 37° C. for 1 hour. The reaction wasstopped by heat inactivation of the terminal transferase at 70° C. for10 min. The resulting product was desalted by passing through a TE-10spin column (Clontech). More than one molecules of biotin-14-dATP couldbe added to the 3′-end of DNA11683. The biotinylated DNA11683 wasincubated with 0.3 mg of Dynal streptavidin beads in 30 μl 1× bindingand washing buffer at ambient temperature for 30 min. The beads werewashed twice with TE and redissolved in 30 μl TE, 10 μl aliquot(containing 0.1 mg of beads) was used for sequencing reactions.

The 0.1 mg beads from previous step were resuspended in a 10 μl volumecontaining 2 μl of 5× Sequenase buffer (200 mM Tris-HCl, pH 7.5, 100 mMMgCl₂, and 250 mM NaCl) from the Sequenase kit and 5 pmol ofcorresponding primer PNA 16/DNA. The annealing mixture was heated to 70°C. and allowed to cool slowly to room temperature over a 20-30 min timeperiod. Then 1 μl 0.1 M dithiothreitol solution, 1 μl Mn buffer (0.15 Msodium isocitrate and 0.1 M MgCl₂), and 2 μl of diluted Sequenase (3.25units) were added. The reaction mixture was divided into four aliquotsof 3 μl each and mixed with termination mixes (each contains of 3 μl ofthe appropriate termination mix: 32 μM c7dATP, 32 μM dCTP, 32 μM c7dGTP,32 μM dTTP and 3.2 μM of one of the four ddTNPs, in 50 mM NaCl). Thereaction mixtures were incubated at 37° C. for 2 min. After thecompletion of extension, the beads were precipitated and the supernatantwas removed. The beads were washed twice and resuspended in TE and keptat 4° C.

Sequencing a 78-Mer Target

Sequencing complex:

(SEQ ID NO: 25) 5′-AAGATCTGACCAGGGATTCGGTTAGCGTGACTGCTGCTGCTGCTGCTGCTGCTGGATGATCCGACGCATCAGATCTGG-(A^(b))_(n)-3′ (TNR.PLASM2) (SEQ ID NO:26) 5′-CTGATGCGTCGGATCATC-3′ (CM1)

The target TNR.PLASM2 was biotinylated and sequenced using proceduressimilar to those described in previous section (sequencing a 39-mertarget).

Sequencing a 15-Mer Target with Partially Duplex Probe

Sequencing complex:

(SEQ ID NO: 27) ^(5′)-F-GATGATCCGACGCATCACAGCTC^(3′) (SEQ ID NO: 28)^(5′)-TCGGTTCCAAGAGCTGTGATGCGTCGGATCATC-b-^(3′)

CM1B3B was immobilized on Dynabeads M280 with streptavidin (Dynal,Norway) by incubating 60 pmol of CM1B3B with 0.3 magnetic beads in 30 μl1 M NaCl and TE (1× binding and washing buffer) at room temperature for30 min. The beads were washed twice with TE and redissolved in 30 μl TE,10 or 20 μl aliquot (containing 0.1 or 0.2 mg of beads respectively) wasused for sequencing reactions.

The duplex was formed by annealing corresponding aliquot of beads fromprevious step with 10 pmol of DF11a5F (or 20 pmol of DF11a5F for 0.2 mgof beads) in a 9 μl volume containing 2 μl of 5× Sequenase buffer (200mM Tris-HCl, pH 7.5, 100 mM MgCl₂₁ and 250 mM NaCl) from the Sequenasekit. The annealing mixture was heated to 65° C. and allowed to coolslowly to 37° C. over a 20-30 min time period. The duplex primer wasthen mixed with 10 pmol of TS10 (20 pmol of TS10 for 0.2 mg of beads) in1 μl volume, and the resulting mixture was further incubated at 37° C.for 5 min, room temperature for 5-10 min. Then 1 μl 0.1 M dithiothreitolsolution, 1 μl Mn buffer (0.15 M sodium isocitrate and 0.1 M MnCl₂), and2 μl of diluted Sequenase (3.25 units) were added. The reaction mixturewas divided into four aliquots of 3 μl each and mixed with terminationmixes (each contains of 4 μl of the appropriate termination mix: 16 μMdATP, 16 μM dCTP, 16 μM dGTP, 16 μM dTTP and 1.6 μM of one of the fourddNTPs, in 50 mM NaCl). The reaction mixtures were incubated at roomtemperature for 5 min, and 37° C. for 5 min. After the completion ofextension, the beads were precipitated and the supernatant was removed.The beads were resuspended in 20 μl TE and kept at 4° C. An aliquot of 2μl (out of 20 μl) from each tube was taken and mixed with 8 μl offormamide, the resulting samples were denatured at 90-95° C. for 5 minand 2 μl (out of 10 μl total) was applied to an ALF DNA sequencer(Pharmacia, Piscataway, N. J.) using a 10% polyacrylamide gel containing7 M urea and 0.6×TBE. The remaining aliquot was used for MALDI-TOF MSanalysis.

MALDI Sample Preparation and Instrumentation

Before MALDI analysis, the sequencing ladder loaded magnetic beads werewashed twice using 50 mM ammonium citrate and resuspended in 0.5 μl purewater. The suspension was then loaded onto the sample target of the massspectrometer and 0.5 μl of saturated matrix solution (3-hydroxypicolinicacid (HPA): ammonium citrate=10:1 mole ratio in 50% acetonitrile) wasadded. The mixture was allowed to dry prior to mass spectrometeranalysis.

The reflectron TOFMS mass spectrometer (Vision 2000, Finnigan MAT,Bremen, Germany) was used for analysis. 5 kV was applied in the ionsource and 20 kV was applied for postacceleration. All spectra weretaken in the positive ion mode and a nitrogen laser was used. Normally,each spectrum was averaged for more than 100 shots and a standard25-point smoothing was applied.

Results And Discussion

Conventional Solid-Phase Sequencing

In conventional sequencing methods, a primer is directly annealed to thetemplate and then extended and terminated in a Sanger dideoxysequencing. Normally, a biotinylated primer is used and the sequencingladders are captured by streptavidin-coated magnetic beads. Afterwashing, the products are eluted from the beads using EDTA andformamide. Previous findings indicated that only the annealed strand ofa duplex is desorbed and the immobilized strand remains on the beads.Therefore, it is advantageous to immobilize the template and anneal theprimer. After the sequencing reaction and washing, the beads with theimmobilized template and annealed sequencing ladder can be loadeddirectly onto the mass spectrometer target and mix with matrix. InMALDI, only the annealed sequencing ladder will be desorbed and ionized,and the immobilized template will remain on the target.

A 39-mer template (SEQ ID NO: 23) was first biotinylated at the 3′-endby adding biotin-14-dATP with terminal transferase. More than onebiotin-14-dATP molecule could be added by the enzyme. Since the templatewas immobilized and remained on the beads during MALDI, the number ofbiotin-14-dATP would not affect the mass spectra. A 14-mer primer (SEQID NO: 24) was used for the solid-state sequencing to generate DNAfragments 3-27 below (SEQ ID NOs: 142-166). MALDI-TOF mass spectra ofthe four sequencing ladders are shown in FIG. 44 and the expectedtheoretical values are shown in Table III.

TABLE III  1   5′-TCTGGCCTGGTGCAGGGCCTATTGTAGTTGTGACGTACA-(A^(B))_(n)-3′  2                            3′-TCAACACTGCATGT-5-  3                           3′-ATCAACACTGCATGT-5′  4                          3′-CATCAACACTGCATGT-5′  5                         3′-ACATCAACACTGCATGT-5′  6                        3′-AACATCAACACTGCATGT-5′  7                       3′-TAACATCAACACTGCATGT-5′  8                      3′-ATAACATCAACACTGCATGT-5′  9                     3′-GATAACATCAACACTGCATGT-5′ 10                    3′-GGATAACATCAACACTGCATGT-5′ 11                   3′-CGGATAACATCAACACTGCATGT-5′ 12                  3′-CCGGATAACATCAACACTGCATGT-5′ 13                 3′-CCCGGATAACATCAACACTGCATGT-5′ 14                3′-TCCCGGATAACATCAACACTGCATGT-5′ 15               3′-GTCCCGGATAACATCAACACTGCATGT-5′ 16              3′-CGTCCCGGATAACATCAACACTGCATGT-5′ 17             3′-ACGTCCCGGATAACATCAACACTGCATGT-5′ 18            3′-CACGTCCCGGATAACATCAACACTGCATGT-5′ 19           3′-CCACGTCCCGGATAACATCAACACTGCATGT-5′ 20          3′-ACCACGTCCCGGATAACATCAACACTGCATGT-5′ 21         3′-GACCACGTCCCGGATAACATCAACACTGCATGT-5′ 22        3′-GGACCACGTCCCGGATAACATCAACACTGCATGT-5′ 23       3′-CGGACCACGTCCCGGATAACATCAACACTGCATGT-5′ 24      3′-CCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′ 25     3′-ACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′ 26    3′-GACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′ 273′-AGACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′ A-reaction C-reactionG-reaction T-reaction  1.  2. 4223.8 4223.8 4223.8 4223.8  3. 4521.1  4.4809.2  5. 5133.4  6. 5434.6  7. 5737.8  8. 6051.1  9. 6379.2 10. 6704.411. 6995.6 12. 7284.8 13. 7574.0 14. 7878.2 15. 8207.4 16. 8495.6 17.8808.8 18. 9097.0 19. 9386.2 20. 9699.4 21. 10027.6 22. 10355.8 23.10644.0 24. 10933.2 25. 11246.4 26. 11574.6 27. 11886.8

The sequencing reaction produced a relatively homogenous ladder, and thefull-length sequence was determined easily. One peak around 5150appeared in all reactions are not identified. A possible explanation isthat a small portion of the template formed some kind of secondarystructure, such as a loop, which hindered sequenase extension.Mis-incorporation is of minor importance, since the intensity of thesepeaks were much lower than that of the sequencing ladders. Although7-deaza purines were used in the sequencing reaction, which couldstabilize the N-glycosidic bond and prevent depurination, minor baselosses were still observed since the primer was not substituted by7-deazapurines. The full length ladder, with a ddA at the 3′ end,appeared in the A reaction with an apparent mass of 11899.8. A moreintense peak of 12333 appeared in all four reactions and is likely dueto an addition of an extra nucleotide by the Sequenase enzyme.

The same technique could be used to sequence longer DNA fragments. A78-mer template containing a CTG repeat (SEQ ID NO: 25) was3′-biotinylated by adding biotin-14-dATP with terminal transferase. An18-mer primer (SEQ ID NO: 26) was annealed right outside the CTG repeatso that the repeat could be sequenced immediately after primerextension. The four reactions were washed and analyzed by MALDI-TOFMS asusual. An example of the G-reaction is shown in FIG. 45 (SEQ ID NOs:167-220) and the expected sequencing ladder is shown in Table IV withtheoretical mass values for each ladder component. All sequencing peakswere well resolved except the last component (theoretical value 20577.4)was indistinguishable from the background. Two neighboring sequencingpeaks (a 62-mer and a 63-mer) were also separated indicating that suchsequencing analysis could be applicable to longer templates. Again, anaddition of an extra nucleotide by the Sequenase enzyme was observed inthis spectrum. This addition is not template specific and appeared inall four reactions which makes it easy to be identified. Compared to theprimer peak, the sequencing peaks were at much lower intensity in thelong template case.

TABLE IvAAGATCTGACCAGGGATTCGGTTAGCGTGACTGCTGCTGCTGCTGCTGGATGATCCGACGCATCAGATCTGG-(A^(B))_(n)-3′ 1                                                      3′-CTACTAGGCTGCGTAGTC-5′ 2                                                     3′-CCTACTAGGCTGCGTAGTC-5′ 3                                                    3′-ACCTACTAGGCTGCGTAGTC-5′ 4                                                   3′-GACCTACTAGGCTGCGTAGTC-5′ 5                                                  3′-CGACCTACTAGGCTGCGTAGTC-5′ 6                                                 3′-ACGACCTACTAGGCTGCGTAGTC-5′ 7                                                3′-GACGACCTACTAGGCTGCGTAGTC-5′ 8                                               3′-CGACGACCTACTAGGCTGCGTAGTC-5′ 9                                              3′-ACGACGACCTACTAGGCTGCGTAGTC-5′10                                             3′-GACGACGACCTACTAGGCTGCGTAGTC-5′11                                            3′-CGACGACGACCTACTAGGCTGCGTAGTC-5′12                                           3′-ACGACGACGACCTACTAGGCTGCGTAGTC-5′13                                          3′-GACGACGACGACCTACTAGGCTGCGTAGTC-5′14                                         3′-CGACGACGACGACCTACTAGGCTGCGTAGTC-5′15                                        3′-ACGACGACGACGACCTACTAGGCTGCGTAGTC-5′16                                       3′-GACGACGACGACGACCTACTAGGCTGCGTAGTC-5′17                                      3′-CGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′18                                     3′-ACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′19                                    3′-GACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′20                                   3′-CGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′21                                  3′-ACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′22                                 3′-GACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′23                                3′-CGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′24                               3′-ACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′25                              3′-GACGACGACGACGACGACGACGACCTACTAGGGTGGGTAGTC-5′26                             3′-TGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′27                            3′-GTGACGAGGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′28                           3′-ACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′29                          3′-CACTGACGACGACGACGACGACGACGACCTAGTAGGCTGCGTAGTC-5′30                         3′-GCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′31                        3′-CGCACTGACGACGACGACGAGGACGACGACCTACTAGGCTGCGTAGTC-5′32                       3′-TCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′33                      3′-ATCGCACTGACGAGGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′34                     3′-AATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′35                    3′-CAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′36                   3′-CCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′37                  3′-GCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′38                 3′-AGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′39                3′-AAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′40               3′-TAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′41              3′-CTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′42             3′-CCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTG-5′43            3′-CCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′44           3′-TCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′45          3′-GTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′46         3′-GGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′47        3′-TGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′48       3′-CTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′49      3′-ACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′50     3′-GACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′51    3′-AGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′52   3′-TAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′53  3′-CTAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′54 3′-TCTAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′553′-TTCTAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′ddATP ddCTP ddGTP ddTTP  1. 5491.6 5491.6 5491.6 5491.6  2. 5764.8  3.6078.0  4. 6407.2  5. 6696.4  6. 7009.6  7. 7338.8  8. 7628.0  9. 7941.210. 8270.4 11. 8559.6 12. 8872.8 13. 9202.0 14. 9491.2 15. 9804.4 16.10133.6 17. 10422.88 18. 10736.0 19. 11065.2 20. 11354.4 21. 11667.6 22.11996.8 23. 12286.0 24. 12599.2 25. 12928.4 26. 13232.6 27. 13521.8 28.13835.0 29. 14124.2 30. 14453.4 31. 14742.6 32. 15046.8 33. 15360.0 34.15673.2 35. 15962.4 36. 16251.6 37. 16580.8 38. 16894.0 39. 17207.2 40.17511.4 41. 17800.6 42. 18189.8 43. 18379.0 44. 18683.2 45. 19012.4 46.19341.6 47. 19645.8 48. 19935.0 49. 20248.2 50. 20577.4 51. 20890.6 52.21194.4 53. 21484.0 54. 21788.2 55. 22092.4

Sequencing Using Duplex DNA Probes for Capturing and Priming

Duplex DNA probes with single-stranded overhang have been demonstratedto be able to capture specific DNA templates and also serve as primersfor solid-state sequencing. The scheme is shown in FIG. 46. Stackinginteractions between a duplex probe and a single-stranded template allowonly a 5-base overhang to be sufficient for capturing. Based on thisformat, a 5′ fluorescent-labeled 23-mer (5′-GAT GAT CCG ACG CAT CAC AGCTC-3′) (SEQ ID NO: 29) was annealed to a 3′-biotinylated 18-mer (5′-GTGATG CGT CGG ATC ATC-3′) (SEQ ID NO: 30), leaving a 5-base overhang. A15-mer template (5′-TCG GTT CCA AGA GCT-3′) (SEQ ID NO: 31) was capturedby the duplex and sequencing reactions were performed by extension ofthe 5-base overhang. MALDI-TOF mass spectra of the reactions are shownin FIG. 47A-D. All sequencing peaks were resolved although at relativelylow intensities. The last peak in each reaction is due to unspecificaddition of one nucleotide to the full length extension product by theSequenase enzyme. For comparison, the same products were run on aconventional DNA sequencer and a stacking fluorogram of the results isshown in FIG. 48. As can be seen from the Figure, the mass spectra hadthe same pattern as the fluorogram with sequencing peaks at much lowerintensity compared to the 23-mer primer.

EXAMPLE 10 Thermo Sequenase Cycle Sequencing Materials and Methods

PCR amplification. Human leukocytic genomic DNA was used for PCRamplification. PCR primers to amplify a 209 bp fragment of the β-globingene were the β2 forward primer (5′-CAT TTG CTT CTG ACA CAA CTG-3′ SEQID NO: 32) and the β11 reverse primer (5′-CTT CTC TGT CTC CAC ATG C-3′SEQ ID NO: 33). Taq polymerase and 10× buffer were purchased fromBoehringer-Mannheim (Germany) and dNTPs from Pharmacia (Freiburg,Germany). The total reaction volume was 50 μl including 8 pmol of eachprimer with approximately 200 ng of genomic DNA used as template and afinal dNTP concentration of 200 μM. PCR conditions were: 5 min at 94°C., followed by 40 cycles of 30 sec at 94° C., 45 sec at 53° C., 30 secat 72° C., and a final extension time of 2 min at 72° C. The generatedamplified product was purified and concentrated (2×) with the Qiagen‘Qiaquick’ PCR purification kit (#28106) and stored in H₂0.

Cycle Sequencing. Sequencing ladders were generated by primer extensionwith Thermo Sequenase™-DNA Polymerase (Amersham LIFE Science, #E79000Y)under the following conditions: 7 pmol of HPLC purified primer (Cod512mer: 5′-TGC ACC TGA CTC-3′ SEQ ID NO: 34) were added to 6 μl purifiedand concentrated amplified product (i.e. 12 μl of the original amplifiedproduct), 2.5 units Thermo Sequenase and 2.5 ml Thermo Sequenasereaction buffer in a total volume of 25 μl. The final nucleotideconcentrations were 30 μM of the appropriate ddNTP (ddATP, ddCTP, ddGTPor ddTTP; Pharmacia Biotech, #27-2045-01) and 210 μM of each dNTP(7-deaza-dATP, DCTP, 7-deaza-GTP, dTTP; Pharmacia Biotech).

Cycling conditions were: denaturation for 4 min at 94° C., followed by35 cycles of 30 sec at 94° C., 30 sec at 38° C., 30 sec at 55° C., and afinal extension of 2 min at 72° C.

Sample preparation and analysis by MALDI-TOF MS. After completion of thecycling program, the reaction volume was increased to 50 μl by additionof 25 μl H₂0. Desalting was achieved by shaking 30 μl of ammoniumsaturated DOWEX (Fluka #44485) cation exchange beads with 50 μl of theanalyte for 2 min at room temperature. The Dowex beads, purchased in theprotonated form, were pre-treated with 2M NH₄0H to convert them to theammonium form, then washed with H₂0 until the supernatant was neutral,and finally put in 10 mM ammonium citrate for usage. After the cationexchange, DNA was purified and concentrated by ethanol precipitation byadding 5 μl 3 M ammonium acetate (pH 6.5), 0.5 μl glycogen (10 mg/ml,Sigma), and 110 μl absolute ethanol to the analyte and incubated at roomtemperature for 1 hour. After 12 min centrifugation at 20,000×g thepellet was washed in 70% ethanol and resuspended in 1 μl 18 Mohm/cm H₂0water.

For MALDI-TOF MS analysis 0.35 μl of resuspended DNA was mixed with0.35-1.3 μl matrix solution (0.7 M 3-hydroxypicolinic acid (3-HPA), 0.07M ammonium citrate in 1:1 H₂0:CH₃CN) on a stainless steel sample targetdisk and allowed to air dry preceding spectrum acquisition using aThermo Bioanalysis Vision 2000 MALDI-TOF operated in reflectron modewith 5 and 20 kV on the target and conversion dynode, respectively.External calibration generated from eight peaks (3000-18000 Da) was usedfor all spectra.

Results

FIG. 49 shows a MALDI-TOF mass spectrum of the sequencing laddergenerated from a biological amplified product as template and a 12mer(5′-TGC ACC TGA CTC-3′ (SEQ ID NO: 34)) sequencing primer. The peaksresulting from depurinations and peaks which are not related to thesequence are marked by an asterisk. MALDI-TOF MS measurements were takenon a reflectron TOF MS. A.) Sequencing ladder stopped with ddATP; B.)Sequencing ladder stopped with ddCTP; C.) Sequencing ladder stopped withddGTP; D.) Sequencing ladder stopped with ddTTP.

FIG. 50 shows a schematic representation of the sequencing laddergenerated in FIG. 49 with the corresponding calculated molecular massesup to 40 bases after the primer (SEQ ID NOs: 221-260). For thecalculation the following masses were used: 3581.4 Da for the primer,312.2 Da for 7-deaza-dATP, 304.2 Da for dTTP, 289.2 Da for dCTP and328.2 Da for 7-deaza-dGTP.

FIG. 51 shows the sequence of the amplified 209 bp amplified productwithin the β-globin gene (SEQ ID NO: 261), which was used as a templatefor sequencing. The sequences of the appropriate PCR primer and thelocation of the 12mer sequencing primer is also shown. This sequencerepresents a homozygote mutant at the position 4 after the primer. In awildtype sequence this T would be replaced by an A.

EXAMPLE 11 Microsatellite Analysis Using Primer Oligo Base Extension(PROBE) and MALDI-TOF Mass Spectrometry Summary

The method uses a single detection primer followed by an oligonucleotideextension step to give products differing in length by a number of basesspecific for the number of repeat units or for second site mutationswithin the repeated region, which can be easily resolved by MALDI-TOFmass spectrometry. The method is demonstrated using as a model systemthe AluVpA polymorphism in intron 5 of the interferon-α receptor genelocated on human chromosome 21, and the poly T tract of the spliceacceptor site of intron 8 from the CFTR gene located on human chromosome7.

Materials and Methods

Genomic DNA was obtained from 18 unrelated individuals and one familyincluding of a mother, father, and three children. The repeated regionwas evaluated conventionally by denaturing gel electrophoresis andresults obtained were confirmed by standard Sanger sequencing.

The primers for PCR amplification (8 pmol each) were IFNAR-IVS5-5′:(5′-TGC TTA CTT AAC CCA GTG TG-3′ SEQ ID NO: 35) and IFNAR-IVS5-3′. 2:(5′-CAC ACT ATG TAA TAC TAT GC-3′ SEQ ID NO: 36) for a part of theintron 5 of the interferon-α receptor gene, and CFEx9-F:(5′-GAA AAT ATCTGA CAA ACT CAT C-3′ SEQ ID NO: 37) (5′-biotinylated) andCFEx9-R:(5′-CAT GGA CAC CAA ATT AAG TTC-3′ SEQ ID NO: 38) for CFTR exon9 with flanking intron sequences of the CFTR gene. Taq-polymeraseincluding 10× buffer were purchased from Boehringer-Mannheim and dNTPswere obtained from Pharmacia. The total reaction volume was 50 μl. PCRconditions were 5 min at 94° C. followed by 40 cycles of: 1 min at 94°C., 45 sec at 53° C., and 30 sec at 72° C., and a final extension timeof 5 min at 72° C.

Amplification products were purified using Qiagen's PCR purification kit(No. 28106) according to manufacturer's instructions. Purified productswere eluted from the column in 50 μl TE-buffer (10 mM Tris-HCl, 1 mMEDTA, pH 7.5).

A) Primer Oligo Base Extension Reaction (Thermo Cycling Method

CyclePROBE was performed with 5 pmol appropriate detection primer(IFN:5′-TGA GAC TCT GTC TC-3′ SEQ ID NO: 39) in a total volume of 25 μlincluding I pmol purified template, 2 units Thermosequenase (AmershamLife Science, Cat. #E79000Y) 2.5 μl Thermosequenase buffer, 25 μmol ofeach deoxynucleotide (7-deaza-dATP, dTTP, and in some experiments extradCTP) and 100 μmol of dideoxyguanine and in some experiments additionalddCTP. Cycling conditions: initial denaturation 94° C. for 5 minfollowed by 30 cycles with 44° C. annealing temperature for 30 sec and55° C. extension temperature for 1 min.

Primer Oligo Base Extension Reaction (Isothermal Method)

10 μl aliquots of the purified double-stranded amplified product (˜3pmol) were transferred to a streptavidin-coated microliter plate well(˜16 pmol capacity per 50 μl volume; No. 1645684 Boehringer-Mannheim),followed by addition of 10 μl incubation buffer (80 mM sodium phosphate,400 mM NaCl, 0.4% Tween 20, pH 7.5) and 30 μl water. After incubationfor 1 hour at room temperature, the wells were washed three times with200 μl washing buffer A (40 mM Tris, 1 mM EDTA, 50 mM NaCl, 0.1% Tween20, pH 8.8) and incubated with 100 μl of 50 mM NaOH for 3 min todenature the double-stranded DNA. Finally, the wells were washed threetimes with 200 μl 70 mM ammonium citrate solution.

The annealing of 100 pmol detection primer (CFpT: 5′-TTC CCC AAA TCCCTG-3′ SEQ ID NO: 40) was performed in 50 μl annealing buffer (50 mMammonium phosphate buffer, pH 7.0 and 100 mM ammonium chloride) at 65°C. for 2 min, at 37° C. for 10 min, and at room temperature for 10 min.The wells were washed three times with 200 μl washing buffer B (40 mMTris, 1 mM EDTA, 50 mM NH₄Cl, 0.1% Tween 20, pH 8.8) and once in 200 μlTE buffer. The extension reaction was performed using some components ofthe DNA sequencing kit from USB (No. 70770) and dNTPs or ddNTPs fromPharmacia. Total reaction volume was 45 μl, containing of 21 μl water, 6μl Sequenase-buffer, 3 μl 100 mM DTT solution, 50 μmol of 7-deaza-dATP,20 μmol ddCTP, 5.5 μl glycerol enzyme dilution buffer, 0.25 μl Sequenase2.0, and 0.25 μl pyrophosphatase. The reaction was pipetted on ice andincubated for 15 min at room temperature and for 5 min at 37° C.Finally, the wells were washed three times with 200 μl washing buffer B.

The extended primer was denatured from the template strand by heating at80° C. for 10 min in 50 μl of a 50 mM ammonium hydroxide solution.

For precipitation, 10 μl 3 M NH4-acetate (pH 6.5), 0.5 μl glycogen (10mg/ml water, Sigma, Cat.#G1765), and 110 μl absolute ethanol were addedto the supernatant and incubated for 1 hour at room temperature. Aftercentrifugation at 13.000 g for 10 min the pellet was washed in 70%ethanol and resuspended in 1 μl 18 Mohm/cm H₂0 water.

Sample preparation was performed by mixing 0.6 μl of matrix solution(0.7 M 3-hydroxypicolinic acid, 0.07 M dibasic ammonium citrate in 1:1H₂0:CH₃CN) with O.3 μl of resuspended DNA/glycogen pellet on a sampletarget and allowed to air dry. Up to 20 samples were spotted on a probetarget disk for introduction into the source region of a ThermoBioanalysis (formerly Finnigan) Visions 2000 MALDI-TOF operated inreflectron mode with 5 and 20 kV on the target and conversion dynode,respectively. Theoretical average molecular mass (M_(r)(calc)) werecalculated from atomic compositions; reported experimental M_(r)(M_(r)(exp)) values are those of the singly-protonated form, determinedusing external calibration.

Results

The aim of the experiments was to develop a fast and reliable method forthe exact determination of the number of repeat units in microsatellitesor the length of a mononucleotide stretch including the potential todetect second site mutations within the polymorphic region. Therefore, aspecial kind of DNA sequencing (primer oligo base extension, PROBE) wascombined with the evaluation of the resulting products bymatrix-assisted laser desorption ionization (MALDI) mass spectrometry(MS). The time-of-flight (TOF) reflectron arrangement was chosen-as apossible mass measurement system. As an initial feasibility study, anexamination was performed first on an AluVpA repeat polymorphism locatedin intron 5 of the human interferon-α receptor gene (cyclePROBEreaction) and second on the poly T tract located in intron 8 of thehuman CFTR gene (isothermal PROBE reaction).

A schematic presentation of the cyclePROBE experiment for the AluVpArepeat polymorphism is given in FIG. 52. The extension of the antisensestrand (SEQ ID NO: 262) was performed with the sense strand serving asthe template. The detection primer is underlined. In a family studyco-dominant segregation of the various alleles could be demonstrated bythe electrophoretic procedure as well as by the cyclePROBE methodfollowed by mass spec analysis (FIG. 53). Those alleles of the motherand child 2, for which direct electrophoresis of the amplified productindicated one of the two copies to have 13 repeat units, were measuredusing cyclePROBE to have instead only 11 units using ddG as terminator.The replacement of ddG by ddC resulted in a further unexpected shortallele with a molecular mass of approximately 11650 in the DNA of themother and child 2 (FIG. 54). Sequence analysis verified this presenceof two second site mutations in the allele with 13 repeat units. Thefirst is a C to T transition in the third repeat unit and the secondmutation is a T to G transversion in the ninth repeat unit. Examinationof 28 unrelated individuals shows that the 13 unit allele is splicedinto a normal allele and a truncated allele using cyclePROBE.Statistical evaluation shows that the polymorphism is in Hardy-Weinbergequilibrium for both methods, however, using cyclePROBE as detectionmethod the polymorphism information content is increased to 0.734.

PROBE was also used as an isothermic method for the detection of thethree common alleles at the intron 8 splice acceptor site of the CFTRgene (SEQ ID NO: 263). FIG. 55 shows a schematic presentation of theexpected diagnostic products (SEQ ID NOs: 264-266) with the theoreticalmass values. The reaction was also performed in the antisense direction.

FIG. 56 demonstrates that all three common alleles (T5, T7, and T9,respectively) at this locus could be reliably disclosed by this method.Reference to FIG. 56 indicates that mass accuracy and precision with thereflectron time of flight used in this study ranged from 0-0.4%, with arelative standard deviation of 0.13%. This corresponds to far betterthan single base accuracy for the up to <90-mer diagnostic productsgenerated in the IFNAR system. Such high analytical sensitivity issufficient to detect single or multiple insertion/deletion mutationswithin the repeat unit or its flanking regions, which would induce >1%mass shifts in a 90-mer. This is analogous to the FIG. 56 polyT tractanalysis. Other mutations (i.e. an A to T or a T to A mutation withinthe IFNAR gene A3T repeat) which do not cause premature producttermination are not detectable using any dNTP/ddNTP combination withPROBE and low performance MS instrumentation; a 9 Da shift in a 90-mercorresponds to a 0.03% mass shift. Achieving the accuracy and precisionrequired to detect such minor mass shifts has been demonstrated withhigher performance instrumentation such as Fourier transform (FT)MS, forwhich single Da accuracy is obtained up to 100-mers. Further, tandemFTMS, in which a mass shifted fragment can be isolated within theinstrument and dissociated to generate sequence specific fragments, hasbeen demonstrated to locate point mutations to the base in comparablysized products. Thus the combination of PROBE with higher performanceinstrumentation will have an analytical sensitivity which can be matchedonly by cumbersome full sequencing of the repeat region.

EXAMPLE 12 Improved Apolipoprotein E Genotyping Using Primer Oligo BaseExtension (PROBE) and MALDI-TOF Mass Spectrometry Materials and Methods

PCR Amplification.

Human leukocytic genomic DNA from 100 anonymous individuals from apreviously published study (Braun, A et al., (1992) Human Genet. 89:401-406) were screened for apolipoprotein E genotypes using conventionalmethods. PCR primers to amplify a portion of exon 4 of the apo E genewere delineated according to the published sequence (Das, H K et al.,(1985) J. Biol. Chem. 260: 6240-6247) (forward primer, apoE-F: 5′-GGCACG GCT GTC CAA GGA G-3′ SEQ ID NO: 41; reverse, apoE-R: 5′-AGG CCG CGCTCG GCG CCC TC-3′ SEQ ID NO: 42). Taq polymerase and 10× buffer werepurchased from Boehringer-Mannheim (Germany) and dNTPs from Pharmacia(Freiburg, Germany). The total reaction volume was 50 μL including 8pmol of each primer and 10% DMSO (dimethylsulfoxide, Sigma) withapproximately 200 ng of genomic DNA used as template. Solutions wereheated to 80° C. before the addition of 1 U polymerase; PCR conditionswere: 2 min at 94° C., followed by 40 cycles of 30 sec at 94° C., 45 secat 63° C., 30 sec at 72° C., and a final extension time of 2 min at 72°C.

Restriction Enzyme Digestion and Polyacrylamide Electrophoresis.

CfoI and RsaI and reaction buffer L were purchased fromBoehringer-Mannheim, and HhaI from Pharmacia (Freiburg, Germany). ForCfoI alone and simultaneous CfoI/RsaI digestion, 20 pL of amplifiedproducts were diluted with 15 μl water and 4 pL Boehringer-Mannheimbuffer L; after addition of 10 units of appropriate restrictionenzyme(s) the samples were incubated for 60 min at 37° C. The procedurefor simultaneous HhaI/RsaI digestion required first digestion by RsaI inbuffer L for one hour followed by addition of NaCl (50 mM endconcentration) and HhaI, and additional incubation for one hour. 20 pLof the restriction digest were analyzed on a 12% polyacrylamide gel asdescribed elsewhere (Hixson (1990) J. Lipid Res. 31: 545-548).Recognition sequences of RsaI and CfoI (HhaI) are GT/AC and GCG/C,respectively; masses of expected digestion fragments from the 252-meramplified product with CfoI alone and the simultaneous double digestwith CfoI (or HhaI) and RsaI are given in Table V.

Thermo-PROBE.

PCR amplification was performed as described above, but with productspurified with the Qiagen ‘Qiaquick’ kit to remove unincorporatedprimers. Multiplex Thermo-PROBE was performed with 35 μl amplifiedproduct and 8 pmol each of the codon 112 (5′-GCG GAC ATG GAG GAC GTG-3′SEQ ID NO: 43) and 158 (5′-GAT GCC GAT GAC CTG CAG AAG-3′ SEQ ID NO: 44)detection primers in 20 μl including ˜1 pmol purified biotinylatedantisense template immobilized on streptavidin coated magnetic beads,2.5 units Thermosequenase, 2 μl Thermosequenase buffer, 50 μM of eachdNTP and 2OO μM of ddXTP, with the base identity of N and X as describedin the text. Cycling conditions were: denaturation (94° C., 30 sec)followed by 30 cycles at 94° C. (10 min) and 60° C. (45 sec).

Sample Preparation and Analysis by MALDI-TOF MS.

For precipitation (Stults et al., (1991) Rapid Commun. Mass Spectrom. 5:359-363) of both digests and PROBE products, 5 μl 3M ammonium acetate(pH 6.5), 0.5 μl glycogen (10 mg/ml, Sigma), and 110 μl absolute ethanolwere added to 50 μl of the analyte solutions and stored for 1 hour atroom temperature. After 10 min centrifugation at 13,000×g the pellet waswashed in 70% ethanol and resuspended in 1 μl 18 Mohm/cm H₂0 water.Where noted in the text, additional desalting was achieved by shaking10-2O μL of ammonium saturated DOWEX (Fluka #44485) cation exchangebeads in 40 μL of analyte. The beads, purchased in the protonated form,were pre-treated with three 5 min spin-decant steps in 2M NH₄0H,followed with H₂0 and 10 mM ammonium citrate.

0.35 μL of resuspended DNA was mixed with 0.35-1.3 μL matrix solutions(Wu et al. (1993) Rapid Commun. Mass Spectrom. 7: 142-146) 0.7 M3-hydroxypicolinic acid (3-HPA), 0.07 M ammonium citrate in1:1H₂0:CH₃CN) on a stainless steel sample target disk and allowed to airdry preceding spectrum acquisition using a Thermo Bioanalysis Vision2000 MALDI-TOF operated in reflectron mode with 5 and 20 kV on thetarget and conversion dynode, respectively. Theoretical averagemolecular masses (M_(r)(calc)) of the fragments were calculated fromatomic compositions; the mass of a proton (1.08 Da) is subtracted fromraw data values in reporting experimental molecular masses (M_(r)(exp))as neutral basis. An external calibration generated from eight peaks(3000-18000 Da) was applied to all spectra.

Results

Digestion with CfoI Alone.

The inset to FIG. 57 a shows a 12% polyacrylamide gel electrophoreticseparation of an ε3/ε3 genotype after digestion of the 252 bp apo Eamplified product with CfoI. Comparison of the electrophoretic bandswith a molecular weight ladder shows the cutting pattern to be as mostlyas expected (Table V) for the ε3/ε3 genotype. Differences are that thefaint band at approximately 25 bp is not expected, and the smallestfragments are not observed. The accompanying mass spectrum ofprecipitated digest products shows a similar pattern, albeit at higherresolution. Comparison with Table V shows that the observed masses areconsistent with those of single-stranded DNA; the combination of anacidic matrix environment (3-HPA, pK_(a) 3) and the absorption ofthermal energy via interactions with the 337 nm absorbing 3-HPA uponionization is known to denature short stretches of dsDNA under normalMALDI conditions (Tang, K et al., (1994) Rapid Commun Mass Spectrom8:183-186).

The approximately 25-mers, unresolved with electrophoresis, are resolvedby MS as three single stranded fragments; while the largest (7427 Da) ofthese may represent a doubly charged ion from the 14.8 kDa fragments(m=14850, z=2; m/z=7425), the 6715 and 7153 Da fragments could resultfrom PCR artifacts or primer impurities; all three peaks are notobserved when amplified products are purified with Qiagen purificationkits prior to digestion. The Table V 8871 Da 29-mer sense strand3′-terminal fragment is not observed; the species detected at 9186 Da isconsistent with the addition of an extra base (9187-8871=316, consistentwith A) by the Taq-polymerase during PCR amplification (Hu, G et al.,(1993) DNA and Cell Biol 12:763-770). The individual single strands ofeach double strand with <35 bases (11 kDa) are resolved as single peaks;the 48-base single strands (M_(r)(calc) 14845 and 14858), however, areobserved as an unresolved single peak at 14850 Da. Separating these intosingle peaks would require a mass resolution (m/

m, the ratio of the mass to the peak width at half height) of14850/13=1140, nearly an order of magnitude greater than what is routinewith the standard reflectron time-of-flight instrumentation used in thisstudy; resolving such small mass differences with high performanceinstrumentation such as Fourier transform MS, which provides up to threeorders of magnitude higher resolution in this mass range, has beendemonstrated. The 91-mer single strands (M_(r)(calc) 27849 and 28436)are also not resolved, even though this requires a resolution of only<50. The dramatic decrease in peak quality at higher masses is due tometastable fragmentation (i.e. depurination) resulting from excessinternal energy absorbed during and subsequent to laser irradiation.

Simultaneous Digestion with CfoI and RsaI.

FIG. 57 b (inset) shows a 12% polyacrylamide gel electrophoresisseparation of ε3/ε3 double digest products, with bands consistent withdsDNA with 24, 31, 36, 48, and 55 base pairs, but not for the smallerfragments. Although more peaks are generated (Table V) than with CfoIalone, the corresponding mass spectrum is more easily interpreted andreproducible since all fragments contain <60 bases, a size range farmore appropriate for MALDI-MS if reasonably accurate M_(r) values (e.g.,0.1%) are desired. For fragments in this mass range, the mass measuringaccuracy using external calibration is −0.1% (i.e. ≦±10 Da at 10 kDa).Significant depurination (indicated in Figure by asterisk) is observedfor all peaks above 10 kDa, but even the largest peak at 17171 Da isclearly resolved from its depurination peak so that an accurate M_(r)can be measured. Although molar concentrations of digest products shouldbe identical, some discrimination against those fragments with ≦11 basesis observed, probably due to their loss in the ethanol/glycogenprecipitation step. The quality of MS results from simultaneousdigestion with CfoI (or HhaI) and RsaI is superior to those with CfoI(or HhaI) alone, since the smaller fragments generated are good forhigher mass accuracy measurements, and with all genotypes there is nopossibility for dimer peaks overlapping with high mass diagnostic peaks.Since digestion by RsaI/CfoI and RsaI/HhaI produce the same restrictionfragments but the former may be performed as a simultaneous digest sincetheir buffer requirements are the same, this enzyme mixture was used forall subsequent genotyping by restriction digest protocols.

TABLE V Mass and Copy Number of Expected Restriction Digest ProductsTable Va Cfol Digestion^(a) (+) (−) e2/e2 e2/e2 e2/e2 e2/e2 e2/e2 e2/e25781, 5999 — — 1 — 1 2 10752, 10921 — 1 1 2 2 2 14845, 14858 — 1 1 2 2 222102, 22440 — — 1 — 1 2 25575, 25763 2 1 1 — — — 27849, 28436 2 2 1 2 1— Table Vb. Cfol/Rsal Digestion^(b) (+) (−) e2/e2 e2/e3 e2/e4 e3/e3e3/e4 e4/e4 3428, 4025 — 1 1 2 2 2 5283, 5880 — — 1 — 1 2 5781, 5999 — —1 — 1 2 11279, 11627 2 2 1 2 1 — 14845, 14858 — 1 1 2 2 2 18269, 18848 22 1 — — — ^(a)Cfol Invariant fragment masses: 1848, 2177, 2186, 2435,4924, 5004, 5412, 5750, 8871, 9628 Da. ^(b)Cfol/Rsal Invariant fragmentmasses: 1848, 2177, 2186, 2436, 4924, 5004, 5412, 5750, 6745, 7510,8871, 9628, 16240, 17175 Da.

TABLE VI ddT M_(r) (Calc) ddT M_(r) (Exp) ddC M₅ (Calc) ddC M_(r) (Exp)e2/e2 ^(a)5918, ^(b)6768      ^(a)6536, ^(b)7387      e2/e3 ^(a)5918,^(b)6768, 5919, 6769, ^(a)6536, ^(b)6753, 6542, 6752, ^(b)7965 7967^(b)7387 7393 e2/e4 ^(a)5918, ^(b)6768,      ^(a)5903, ^(b)6536,     ^(b)7965, ^(a)8970 ^(b)6753, ^(a)7387 e3/e3 ^(a)5918, ^(b)7965 5918,7966 ^(a)6536, ^(b)6753 6542, 6756 e3/e4 ^(a)5918, ^(b)7965, 5914, 7959,^(a)5903, ^(b)6536, 5898, 6533, ^(a)8970 8965 ^(b)6753 6747 e4/e4^(b)7965, ^(a)8970 7966, 8969 ^(a)5903, ^(b)6753 5900, 6752 ^(a)Fromcodon 112 detection primer (unextended 5629.7 Da). ^(b)From codon 158detection primer (unextended 6480.3 Da). Dashed lines: this genotype notavailable from the analyzed pool of 100 patients.

FIG. 58 a-c shows the ApoE ε3/ε3 genotype after digestion with CfoI anda variety of precipitation schemes; equal volume aliquots of the sameamplified product were used for each. The sample treated with a singleprecipitation (FIG. 58 a) from an ammonium acetate/ethanol/glycogensolution results in a mass spectrum characterized by broad peaks,especially at high mass. The masses for intense peaks at 5.4, 10.7, and14.9 kDa are 26 Da (0.5%), 61 Da (0.6%), and 45 Da (0.3%) Da higher,respectively, than the expected values; the resolution (the ratio of apeak width at half its total intensity to the measured mass of the peak)for each of these is −50, and decreases with increasing mass. Suchobservations are consistent with a high level of nonvolatile cationadduction; for the 10.8 kDa fragment, the observed mass shift isconsistent with a greater than unit ratio of adducted:nonadductedmolecular ions.

MS peaks from a sample redissolved and precipitated a second time arefar sharper (FIG. 58 b), with resolution values nearly double those ofthe corresponding FIG. 58 a peaks. Mass accuracy values are alsoconsiderably improved; each is within 0.07% of its respective calculatedvalues, close to the independently determined instrumental limits forDNA measurement using 3-HPA as a matrix. Single (not shown) and double(FIG. 58C) precipitations with isopropyl alcohol (IPA) instead ofethanol result in resolution and mass accuracy values comparable tothose for corresponding ethanol precipitations, but enhanced levels ofdimerization are observed, again potentially confusing measurements whensuch dimers overlap with higher mass “diagnostics” monomers present inthe solution. EtOH/ammonium acetate precipitation with glycogen as anucleation agent results in nearly quantitative recovery of fragmentsexcept for the 7-mers, serving as a simultaneous concentration anddesalting step prior to MS detection. Precipitation from the sameEtOH/ammonium acetate solutions in the absence of glycogen results infar poorer recovery, especially at low mass.

The results indicate that to obtain accurate (M_(r)(exp) values aftereither 1 PA and EtOH precipitations, a second precipitation is necessaryto maintain high mass accuracy and resolution.

The ratio of matrix:digest product also affects spectral quality; severesuppression of higher mass fragments (not shown) observed with 1:1volume matrix: digest product (redissolved in 1 μL) is alleviated byusing a 3-5 fold volume excess of matrix.

Apo E genotyping by enzymatic digestion. Codon 112 and 158 polymorphismsfall within CfoI (but not RsaI) recognition sequences. In the 252 bpamplified product studied here, invariant (i.e. cut in all genotypes)sites cause cuts after bases 31, 47, 138, 156, 239, and 246. The cuttingsite after base 66 is only present for ε4, while that after base 204 ispresent in ε3 and ε4; the ε2 genotype is cut at neither of these sites.These differences in the restriction pattern can be demonstrated asvariations in mass spectra. FIG. 59 shows mass spectra from several ApoEgenotypes available from a pool of 100 patients (Braun, A et al., (1992)Hum. Genet. 89: 401-406). Vertical dashed lines are drawn through thosemasses corresponding to the expected Table V diagnostic fragments; otherlabeled fragments are invariant. Referring to Table V, note that afragment is only considered “invariant” if it is present in duplicatecopies for a given allele; to satisfy this requirement, such a fragmentmust be generated in each of the ε2m ε3, and ε4 alleles.

The spectrum in FIG. 59 a contains all of the expected invariantfragments above 3 kDa, as well as diagnostic peaks at 3428 and 4021(both weak), 11276 and 11627 (both intense), 14845, 18271, and 18865 Da.The spectrum in FIG. 59 b is nearly identical except that the pair ofpeaks at 18 kDa is not detected, and the relative peak intensities, mostnotably among the 11-18 kDa fragments, are different. The spectrum inFIG. 59 c also has no 18 kDa fragments, but instead has new lowintensity peaks between 5-6 kDa. The intensity ratios for fragmentsabove 9 kDa are similar to those of FIG. 59 b except for a relativelylower 11 kDa fragment pair. FIG. 59 d, which again contains the 5-6 kDacluster of peaks, is the only spectrum with no 11 kDa fragments, andlike the previous two also has no 18 kDa fragment.

Despite the myriad of peaks in each spectrum, each genotype can beidentified by the presence and absence of only a few of the Table Vbdiagnostic peaks. Due to the limited resolution of the MALDI-TOFinstrumentation employed, the most difficult genotypes to differentiateare those based upon the presence or absence of the four diagnosticfragments between 5.2 and 6.0 kDa characteristic of the ε4 allele, sincethese fragments nearly overlap with several invariant peaks. It has beenfound herein that the 5283 Da diagnostic fragment overlaps with adepurination peak from the 5412 Da invariant fragment, and the 5781 Dadiagnostic peak is normally not completely resolved from the 5750 Dainvariant fragment. Thus, distinguishing between an ε2/ε4 and ε2/ε3, orbetween an ε3/ε4 and an ε3/ε3 allele, relies upon the presence orabsence of the 5880 and 5999 Da fragments. Each of these is present inFIGS. 59 c and 59 d, but not in 59 a or 59 b.

The genotype of each of the patients in FIG. 59 can be more rapidlyidentified by reference to the flowchart in FIG. 60. Consider the FIG.59 a spectrum. The intense pair of peaks at 11 kDa discounts thepossibility of homozygous ε4, but does not differentiate between theother five genotypes. Likewise, the presence of the unresolved 14.8 kDafragments is inconsistent with homozygous ε2, but leaves fourpossibilities (ε2/ε3, ε2/ε4, ε3/ε3, ε3/ε4). Of these only ε2/ε3 andε2/ε4 are consistent with the 18 kDa peaks; the lack of peaks at 5283,5879, 5779, and 5998, Da indicate that the FIG. 59 a sample is ε2/ε3.Using the same procedure, the FIGS. 59 b-d genotypes can be identifiedas ε3/ε3, ε3/ε4, and ε4/ε4, respectively. To date, all alleleidentifications by this method have been consistent with, and in manycases more easily interpreted than, those attained via conventionalmethods. The assignment can be further confirmed by assuring thatfragment intensity ratios are consistent with the copy numbers of TableV. For instance, the 14.8 kDa fragments are of lower intensity thanthose at 16-17 kDa in FIG. 59 a, but the opposite is seen in FIGS. 59b-d. This is as expected, since in the latter three genotypes the 14.8kDa fragments are present in duplicate, but the first is a heterozygotecontaining ε2, so that half of the amplified products do not contributeto the 14.8 kDa signal. Likewise, comparison of the 11 kDa fragmentintensify to those at 9.6 and 14.8 kDa indicate that this fragment isdouble, double, single, and zero copy in FIGS. 59 a, d, respectively.These data confirm that MALDI can perform in a semi-quantitative wayunder these conditions.

ApoE genotyping by Primer Oligo Base Extension (PROBE). The PROBEreaction was also tested as a means of simultaneous detection of thecodon 112 and 158 polymorphisms. A detection primer is annealed to asingle-stranded PCR-amplified template so that its 3′ terminus is justdownstream of the variable site. Extension of this primer by a DNApolymerase in the presence of three dNTPs and one ddXTP (that is notpresent as a dNTP) results in products whose length and mass depend uponthe identity of the polymorphic base. Unlike standard Sanger typesequencing, in which a particular base-specific tube contains −99% dXTPand −1% ddXTP, the PROBE mixture contains 100% of a particular ddXTPcombined with the other three dNTPs. Thus with PROBE a full stop of alldetection primers is achieved after the first base complementary to theddXTP is reached.

For the ε2/ε3 genotype, the PROBE reaction (mixture of ddTTP, dATP,dCTP, dGTP) causes a M_(r)(exp) shift of the codon 112 primer to 5919Da, and of the codon 158 primer to 6769 and 7967 Da (Table VI); a pairof extension products results from the single codon 158 primer becausethe ε2/ε3 genotype is heterozygous at this position. Three extensionproducts (one from codon 158, two from 112) are also observed from theheterozygote ε3/ε4 (FIG. 61 c and Table VI), while only two products(one from each primer) are observed from the FIG. 61 b (ε3/ε3) and FIG.59 d (ε4/ε4) homozygote alleles. Referring to Table VI, each of theavailable alleles result in all expected ddT reaction product masseswithin 0.1% of the theoretical mass, and thus each is unambiguouslycharacterized by this data alone. Further configuration of the alleleidentities may be obtained by repeating the reaction with ddCTP (plusdATP, dTTP, dGTP); these results, summarized also in Table VI,unambiguously confirm the ddT results.

Appropriateness of the methods. Comparison of FIGS. 59 (restrictiondigestion) and 61 (PROBE) indicates that the PROBE method provides farmore easily interpreted spectra for the multiplex analysis of codon 112and 158 polymorphisms than does the restriction digest analysis. Whilethe digests generate up to −25 peaks per mass spectrum and in some casediagnostic fragments overlapping with invariant fragments, the PROBEreaction generates a maximum of only two peaks per detection primer(i.e. polymorphism). Automated peak detection, spectrum analysis, andallele identification would clearly be far more straightforward for thelatter. Spectra for highly multiplexed PROBE, in which severalpolymorphic sites from the same or different amplified products aremeasured from one tube, are also potentially simple to analyze.Underscoring its flexibility, PROBE data analysis can be furthersimplified by judicious a priori choice of primer lengths, which can bedesigned so that no primers or products can overlap in mass.

Thus while PROBE is the method of choice for large scale clinicaltesting of previously well characterized polymorphic sites, therestriction digest analysis as described here is ideally suited toscreening for new mutations. The identity of each of the twopolymorphisms discussed in this study affects the fragment pattern; ifthis is the only information used, then the MS detection is a fasteralternative to conventional electrophoretic separation of restrictionfragment length polymorphism products. The exact measurement of fragmentM_(r) values can also give information on about sites completely remotefrom the enzyme recognition site since other single point mutationsnecessarily alter the mass of each of the single strands of the doublestranded fragment containing the mutation. The 252 bp amplified productcould also contain allelic variants resulting in, for example,previously described GlyI27 Asp (Weisgraber, K H et al., (1984) J. Clin.Invest. 73: 1024-1033), ArgI36Ser (Wardell, M R et al., (1987) J. Clin.Invest. 80: 483-490), ArgI42Cys (Horie, Y et al., (1992) J. Biol. Chem.267: 1962-1968), Arg145Cys (Rall S C Jr et al., (1982) Proc. Natl. Acad.Sci. U.S.A. 79: 4696-4700), LysI46Glu (Mann, W A et al., (1995) J. Clin.Invest. 96: 1100-1107), or LysI46Gln (Smit, M et al., (1990) J. LipidRes. 31: 45-53) substitutions. The G→A base substitution which codes forthe Gly127 Asp amino acid substitution would result in a −16 Da shift inthe sense strand, and in a +15 Da (C→T) shift in the antisense strand,but not in a change in the restriction pattern. Such a minor changewould be virtually invisible by electrophoresis; however, with accuratemass determination the substitution could be detected; the invariant55-mer fragment at 16240 (sense) and 17175 Da would shift to 16224 and17190 Da, respectively. Obtaining the mass accuracy required to detectsuch minor mass shifts using current MALDI-TOF instrumentation, evenwith internal calibration, is not routine since minor unresolved adductsand/or poorly defined peaks limit the ability for accurate mass calling.With high performance electrospray ionization Fourier transform(ESI-FTMS) single Da accuracy has been achieved with syntheticoligonucleotides (Little, D P et al., (1995) Proc. Natl. Acad. Sci.U.S.A. 92: 2318-2322) up to 100-mers (Little, D P et al., (1994) J. Am.Chem. Soc. 116: 4893-4897), and similar results have recently beenachieved with up to 25-mers using MALDI-FTMS (Li, Y et al., (1996) Anal.Chem. 68: 2090-2096).

EXAMPLE 13 A Method for Mass Spectrometric Detection of DNA FragmentsAssociated With Telomerase Activity Introduction

One-fourth of all deaths in the United States are due to malignanttumors (R. K. Jain, (1996) Science 271:1079-1080). For diagnostic andtherapeutic purposes there is a high interest in reliable and sensitivemethods of tumor cell detection.

Malignant cells can be distinguished from normal cells by differentproperties. One of those is the immortalization of malignant cells whichenables uncontrolled cell-proliferation. Normal diploid mammalian cellsundergo a finite number of population doublings in culture, before theyundergo senescence. It is supposed that the number of populationdoublings in culture, before they undergo senescence. It is supposedthat the number of population doublings is related to the shortening ofchromosome ends, called telomers, in every cell division. The reason forsaid shortening is based on the properties of the conventionalsemiconservative replication machinery. DNA polymerases only work in 5′to 3′ direction and need an RNA primer.

Immortalization is thought to be associated with the expression ofactive telomerase. Said telomerase is a ribonucleoprotein catalyzingrepetitive elongation of templates. This activity can be detected in anative protein extract of telomerase containing cells by a specialPCR-system (N. W. Kim et al. (1994) Science 266: 2011-2015) known astelomeric repeat amplification protocol (TRAP). The assay, as usedherein, is based on the telomerase specific extension of a substrateprimer (TS) and a subsequent amplification of the telomerase specificextension products by a PCR step using a second primer (bioCX)complementary to the repeat structure. The characteristic ladderfragments of those assays are conventionally detected by the use of gelelectrophoretic and labeling or staining systems. These methods can bereplaced by MALDI-TOF mass spectrometry leading to faster accurate andautomated detection.

Materials and Methods

Preparation of Cells

1×10⁶ cultured telomerase-positive cells were pelleted, washed once withPBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄.7H₂O, 1.4 mM KH₂PO₄ insterile DEPC water). The prepared cells may be stored at −75° C. Tissuesamples have to be homogenized, according to procedures well known inthe art, before extraction.

Telomerase Extraction

Pellet was resuspended in 200 μl CHAPS lysis buffer (10 mM Tris-HCl pH7.5, 1 mM MgCl₂, 1 mM EGTA, 0.1 mM benzamidine, 5 mM β-mercaptoethanol,0.5% CHAPS, 10% glycerol) and incubated on ice for 30 min. The samplewas centrifuged at 12,000 g for 30 min at 4° C. The supernatant wastransferred into a fresh tube and stored at 75° C. until use.

TRAP-Assay

2 μl of telomerase extract were added to a mixture of 10×TRAP buffer(200 mM Tris-HCl pH 8.3, 15 mM MgCl₂, 630 mM KCl, 0.05% Tween 20, 10 mMEGTA) 50×dNTP-mix (2.5 mM each dATP, dTTP, dGTP, and dCTP), 10 pmol ofTS primer and 50 pmol of bio CX primer in a final volume of 50 μl. Themixture was incubated at 30° C. for 10 minutes and 5 min. at 94° C., 2units of Taq Polymerase were added and a PCR was performed with 30cycles of 94° C. for 30 seconds, 50° C. for 30 seconds and 72° C. for 45seconds.

Purification of TRAP-Assay Products

For every TRAP-assay to be purified, 50 μl Streptavidin M-280 Dynabeads(10 mg/ml) were washed twice with 1×BW buffer (5 mM Tris-HCl, pH 7.5,0.5 mM EDTA, 1 M NaCl). 50 μl of 2×BW buffer were added to the PCR mixand the beads were resuspended in this mixture. The beads were incubatedunder gentle shaking for 15 min. at ambient temperature. The supernatantwas removed and the beads were washed twice with 1×BW buffer. To thebeads 50 μl 25% ammonium hydroxide were added and incubated at 60° C.for 10 min. The supernatant was saved, the procedure repeated, bothsupernatants were pooled and 300 μl ethanol (100%) were added. After 30min. the DNA was pelleted at 13,000 rpm for 12 min., the pellet wasair-dried and resuspended in 600 nl ultrapure water.

MALDI-TOF MS of TRAP-Assay Products

300 nl sample were mixed with 500 nl of saturated matrix-solution(3-HPA:ammonium citrate=10:1 molar ratio in 50% aqueous acetonitrile),dried at ambient temperature and introduced into the mass spectrometer(Vision 2000, Finigan MAT). All spectra were collected in reflector modeusing external calibration.

Sequences and Masses

bioCX: d(bio-CCC TTA CCC TTA CCC TTA CCC TAA SEQ ID NO: 45), mass: 7540Da. TS: d(AAT CCG TGC AGC AGA GTT SEQ ID NO: 46), mass: 5523 Da.Telomeric-repeat structure: (TTAGGG)_(n), mass of one repeat: 1909.2Amplification products:TS elongated by three telomeric repeats (first amplification product):12452 Da. (N₃)TS elongated by four telomeric repeats: 14361 Da. (N₄)TS elongated by seven telomeric repeats: 20088 Da. (N₇)

Results

FIG. 62 depicts a section of a TRAP-assay MALDI-TOF mass spectrum.Assigned are the primers TS and bioCX at 5497 and 7537 Da, respectively(calculated 5523 and 7540 Da). The signal marked by an asteriskrepresents n-1 primer product of chemical DNA synthesis. The firsttelomerase specific TRAP-assay product is assigned at 12775 Da. Thisproduct represents a 40-mer containing three telomeric repeats. Due toprimer sequences this is the first expected amplification product of apositive TRAP-assay. The product is elongated by an additionalnucleotide due to extendase activity of Taq DNA polymerase (calculatednon-extended product: 12452 Da, by A extended product: 12765 Da). Thesignal at 6389 Da represents the doubly charged ion of this product(calculated: 6387 Da). FIG. 63 shows a section of higher masses of thesame spectrum as depicted in FIG. 62, therefore the signal at 12775 Dais identical to that in FIG. 62. The TRAP-assay product containing seventelomeric repeats, representing a 64-mer also elongated by an additionalnucleotide, is detected at 20322 Da (calculated: 20395 Da). The signalsmarked 1, 2, 3 and 4 cannot be base-line resolved. This region includesof: 1. signal of dimeric n-1 primer, 2. second TRAP-assay amplificationproduct, containing 4 telemetric repeats and therefore representing a46-mer (calculated: 14341 Da/14674 Da for extendase elongated product)and 3. dimeric primer-ion and furthermore all their correspondingdepurination signals. There is a gap observed between the signals of thesecond and fifth extension product. This signal gap corresponds to thereduced band intensities observed in some cases for the third and fourthextension product in autoradiographic analysis of TRAP-assays (N. W. Kimet al. (1994) Science 266: 2013).

The above-mentioned problems, caused by the dimeric primer and relatedsignals, can be overcome using an ultrafiltration step employing amolecular weight cut-off membrane for primer removal prior toMALDI-TOF-MS analysis. This will permit an unambiguous assignment of thesecond amplification product.

EXAMPLE 14 A Method for Detecting Neuroblastoma-Specific NestedRT-Amplified Products Via MALDI-TOF Mass Spectrometry Introduction

Neuroblastoma is predominantly a tumor of early childhood with 66% ofthe cases presenting in children younger than 5 years of age. The mostcommon symptoms are those due to tumor mass, bone pain, or those causedby excessive catecholamine secretion. In rare cases, neuroblastoma canbe identified prenatally (R. W. Jennings et al, (1993) J. Ped. Surgery28: 1168-1174). Approximately 70% of all patients with neuroblastomahave metastatic disease at diagnosis. The prognosis is dependent on ageat diagnosis, clinical stage and other parameters.

For diagnostic purposes there is a high interest in reliable andsensitive methods of tumor cell detection, e.g., in control ofautologous bone marrow transplants or on-going therapy.

Since catecholamine synthesis is a characteristic property ofneuroblastoma cells and bone marrow cells lack this activity (H. Naitoet al., (1991) Eur. J. Cancer 27: 762-765), neuroblastoma cells ormetastasis in bone marrow can be identified by detection of humantyrosine 3-hydroxylase (E. C. 1.14.16.2, hTH) which catalyzes the firststep in biosynthesis of catecholamines.

The expression of hTH can be detected via reverse transcription (RT)polymerase chain reaction (PCR) and the amplified product can beanalyzed via MALDI-TOF mass spectrometry.

Materials and Methods

Cell- or Tissue-Treatment

Cultures cells were pelleted (10 min. 8000 rpm) and washed twice withPBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄.7H₂O, 1.4 mM KH₂PO₄ insterile PEPC water). The pellet was resuspended in 1 ml lysis/bindingbuffer (100 mM Tris-HCl, pH 8.0, 500 mM LiCl, 10 mM EDTA, 1% Li-dodecylsulfate, 5 mM DTT) until the solution becomes viscose. Viscosity wasreduced by DNA-shear step using a 1 ml syringe. The lysate may be storedin −75° C. or processed further directly. Solid tissues (e.g., patientsamples) have to be homogenized before lysis.

Preparation of Magnetic Oligo-dT(25) Beads

100 μL beads per 1×10⁶ cells were separated from the storage buffer andwashed twice with 200 μL lysis/binding buffer.

Isolation of Poly A⁺ RNA

The cell lysate was added to the prepared beads and incubated for 5 min.at ambient temperature. The beads were separated magnetically for 2-5min. and washed twice with 0.5 ml LDS (10 mM Tris-HCl, pH 8.0, 0.15 MLiCl, 1 mM EDTA, 0.1% LiDS).

Solid-Phase First-Strand cDNA Synthesis

The poly A⁺ RNA containing beads were resuspended in 20 μL of reversetranscription mix (50 mM Tris-HCl, pH 8.3, 8 mM MgCl₂, 30 mM KCl, 10 mMDTT, 1.7 mM dNTPs, 3 U AMV reverse transcriptase) and incubated for 1hour at 45° C. (with a resuspension step all ten min.). The beads wereseparated from the reverse transcription mix, resuspended in 50 μL ofelution buffer (2 mM EDTA pH 8.0) and heated to 95° C. for 1 min. furelution of the RNA. The beads with the cDNA first-strand can be storedin TB (0.089 M Tris-base, 0.089 M boric acid, 0.2 mM EDTA pH 8.0), TE 10mM Tris-HCl, 0.1 mM EDTA, pH 8.0) or 70% ethanol for further processing.

Nested Polymerase Chain Reaction

Beads containing cDNA first-strand were washed twice with 1×PCR buffer(20 mM Tris-HCl pH 8.75, 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO₄, 0.1%Triton X-100, 0.1 mg bovine serum albumin) and resuspended in PCR mix(containing 100) pmol of each outer primer, 2.5 u Pfu (exo−) DNApolymerase, 200 μM of each dNTP and PCR buffer in a final volume of 50μL). The mixture was incubated at 72° C. 1 min. and amplified by PCR for30 cycles. for the nested reaction: 1 μL of the first PCR was added astemplate to a PCR mix d (as above but nested primers instead of outerprimers) and subjected to the following temperature program: 94° C. 1min., 65° C. 1 min. and 72° C. 1 min. for 20 cycles.

Purification of Nested Amplified Products

Primers and low-molecular reaction by-products are removed using 10,000Da cut-off ultrafiltration-unit. Ultrafiltration was performed at 7,500g for 25 minutes. For every PCR to be purified, 50 μL Streptavidin M-280Dynabeads (10 mg/ml) were washed twice with 1×BW buffer (5 mM Tris-HCl,pH 7.5, 0.5 mM EDTA, 1 M NaCl), added to the ultrafiltration membraneand incubated under gentle shaking for 15 min. at ambient temperature.The supernatant was removed and the beads were washed twice with 1×BWbuffer. 50 μL 25% ammonium hydroxide were added to the beads andincubated at ambient temperature for 10 min. The supernatant was saved,the procedure repeated, both supernatants were pooled and 300 μL ethanol(100%) were added. After 30 min. the DNA was pelleted at 13,000 rpm for12 min., the pellet was air-dried and resuspended in 600 nl ultrapurewater.

MALDI-TOF MS of Nested Amplified Products

300 nl sample was mixed with 500 nl of saturated matrix-solution (3-HPA:ammonium citrate=10:1 molar ratio in 50% aqueous acetonitrile), dried atambient temperature and introduced into the mass spectrometer (Vision2000, Finigan MAT). All spectra were collected in reflector mode usingexternal calibration.

Outer primers:

hTH1: d(TGT CAG AGC TGG ACA AGT GT SEQ ID NO: 47) hTH2: d(GAT ATT GTCTTC CCG GTA GC SEQ ID NO: 48) Nested primers: bio-hTH d(bio-CTC GGA CCAGGT GTA CCG CC SEQ ID NO: 49), mass: 6485 Da hTH6; d(CCT GTA CTG GAA GGCGAT CTC SEQ ID NO: 50), mass: 6422 21 Da

-   -   mass of biotinylated single strand amplified product: 19253:6 Da    -   mass of nonbiotinylated single strand amplified product: 18758.2        Da

Results

A MALDI-TOF mass spectrum of a human tyrosine 3-hydroxylase (hTH)specific nested amplified product (61-mer) is depicted in FIG. 64. Thesignal at 18763 Da corresponds to non-biotinylated strand of theamplified product (calculated: 18758.2 Da, mass error: 0.02 Da). Thesignals below 10,000 and above 35,000 Da are due to multiply charged anddimeric amplified product-ions, respectively.

The product was obtained from a solid phase cDNA derived in a reversetranscription reaction from 1×10⁶ cells of a neuroblastoma cell-line(L-A-N-1) as described above. The cDNA first-strand was subjected to afirst PCR using outer primers (hTH1 and hTH2), an aliquot of this PCRwas used as template in a second PCR using nested primers (biohTH andhTH6). The nested amplified product was purified and MALDI-TOF MSanalyzed:

The spectrum in FIG. 64 demonstrates the possibility of neuroblastomacell detection using nested RT-PCR and MALDI-TOF MS analysis.

EXAMPLE 15 Rapid Detection of the RET Proto-Oncogene Codon 634 MutationUsing Mass Spectrometry Material and Methods

Probe

The identity of codon 634 in each of the three alleles was confirmed byRsaI enzymatic digestion, single strand conformational polymorphism orSanger sequencing. Exon 11 of the RET gene was PCR amplified (40 cycles)from genomic DNA using Taq-Polymerase (Boehringer-Mannheim) with 8 pmoleach of 5′-biotinylated forward (5′-biotin-CAT GAG GCA GAG CAT ACG CA-3′SEQ ID NO: 51) and unmodified reverse (5′-GAC AGC AGC ACC GAG ACG AT-3′SEQ ID NO: 52) primer per tube; amplified products were purified usingthe Qiagen (QIAquick” kit to remove unincorporated primers. 15 μl ofamplified product were immobilized on 10 μL (10 mg/mL) Dynalstreptavidin coated magnetic beads, denatured using the manufacturer'sprotocol, and the supernatant containing antisense strand discarded, thePROBE reaction was performed using thermoSequenase (TS) DNA Polymerase(Amersham) and Pharmacia dNTP/ddNTPs. 8 pmol of extension primer (5′-CGGCTG CGA TCA CCG TGC GG-3′ SEQ ID NO: 53) was added to 13 μL H₂O, 2 μLTS-buffer, 2 μL 2 mM ddATP (or ddTTP), and 2 μL of 0.5 mM dGTP/dCTP/dTTP(or dGTP/DCTP/dATP), and the mixture heated for 30 sec @ 94° C.,followed by 30 cycles of 10 sec @ 94° C. and 45 sec @ 50° C.; after a 5min. incubation @ 95° C., the supernatant was decanted, and productswere desalted by ethanol precipitation with the addition of 0.5 μL of 10mg/mL glycogen. The resulting pellet was washed in 70% ethanol, airdried, and suspended in 1 μL H₂O. 300 nL of this was mixed with theMALDI matrix (0.7 M 3-hydroxypicolinic acid, 0.07 M ammonium citrate in1:1 H₂O:CH₃CN) on a stainless steel sample probe and air dried. Mass,spectra were collected on a Thermo Bionalysis Vision 2000 MALDI-TOFoperated in reflectron mode with 5 and 20 kV on the target andconversion dynode, respectively. Experimental masses (m_(r)(exp))reported are those of the neutral molecules as measured using externalcalibration.

Direct Measurement of Diagnostic Products

PCR amplifications conditions for a 44 bp region containing codon 634were the same as above but using Pfu polymerase; the forward primercontained a ribonucleotide at its 3′-terminus (forward, 5′-GAT CCA CTGTGC GAC GAG C (SEQ ID NO: 54)-ribo; reverse, 5′-GCG GCT GCG ATC ACC GTGC (SEQ ID NO: 55). After product immobilization and washing, 80 μL of12.5% NH₄0H was added and heated at 80° C. overnight to cleave theprimer from 44-mer (sense strand) to give a 25-mer. Supernatant waspipetted off while still hot, dried resuspended in 50 μL H₂O,precipitated, resuspended, and measured by MALDI-TOF as above.MALDI-FTMS spectra of 25-mer synthetic analogs were collected aspreviously described (Li, Y. et al., (1996) Anal. Chem. 68: 2090-2096);briefly, 1-10 pmol DNA was mixed 1:1 with matrix on a direct insertionprobe, admitted into the external ion source (positive ion mode),ionized upon irradiance with a 337 nm wavelength laser pulse, andtransferred via rf-only quadruple rods into a 6.5 Tesla magnetic fieldwhere they were trapped collisionally. After a 15 second delay, ionswere excited by a broadband chirp pulse and detected using 256K datapoints, resulting in time domain signals of 5 s duration. Reported(neutral) masses are those of the most abundant isotope peak aftersubtracting the mass of the charge carrying proton (1.01 Da).

Results

The first scheme presented utilizes the PROBE reaction shownschematically in FIG. 65. A 20-mer primer is designed to bindspecifically to a region on the complementary template downstream of themutation site; upon annealing to the template, which is labelled withbiotin and immobilized to streptavidin coated magnetic beads, the PROBEprimer is presented with a mixture of the three deoxynucleotidetriphosphates (dNTPs), a di-dNTP (ddNTP), and a DNA polymerase (FIG.65). The primer is extended by a series of bases specific to theidentity of the variable base in codon 634; for any reaction mixture(e.g., ddA+dT+dC+dG), three possible extension products representing thethree alleles are possible (FIG. 65).

For the negative control (FIG. 66), the PROBE reaction with ddATP+dNTPs(N=T, C, G) causes a M_(r)(exp) shift of the primer from 6135 to 6726 Da(Δm+591). The absence of a peak at 6432 rules out a C→A mutation (FIG.65); the mass of the single observed peak is more consistent withextension by C-ddA (M_(r)(calc) 6721, +0.07% error) than by T-ddA(Mr(calc) 6736, −0.15% error) than of A₃TC₂G expected for C→A mutant.Combining the ddA and ddT reaction data, it is clear that the negativecontrol is as expected homozygous normal at codon 634.

The ddA reaction for patient 1 also results in a single peak(M_(r)(exp)=6731) between expected values for wildtype and C→T mutation(FIG. 65 b). The ddT reaction, however, results in two clearly resolvedpeaks consistent with a heterozygote wildtype (M_(r)(exp) 8249, +0.04%mass error)/C→T mutant (M_(r)(exp) 6428 Da, +0.08% mass error). Forpatient 2, the pair of FIG. 66 c ddA products represent a heterozygoteC→A (M_(r)(exp) 6431, −0.06% mass error)/normal (M_(r)(exp) 6719, −0.03%mass error) allele. The ddT reaction confirms this, with a single peakmeasured at 8264 Da consistent with unresolved wildtype and C→A alleles.The value of duplicate experiments is seen by comparing FIGS. 66 a and66 b; while for patient 1 the peak at 6726 from the ddA reactionrepresents only one species, similar peak from patient 1 is actually apair of unresolved peaks differing in mass by 15 Da.

An alternate scheme for point mutation detection is differentiation ofalleles by direct measurement of diagnostic product masses. A 44-mercontaining the RET634 site was generated by the PCR, and the 19-mersense primer removed by NH₄0H cleavage at a ribonucleotide at its 3′terminus.

FIG. 67 shows a series of MALDI-FTMS spectra of synthetic analogs ofshort amplified products containing the RET634 mutant site. FIGS. 67 a-cand 67 d-f are homozygous and heterozygous genotypes, respectively. Aninternal calibration was done using the most abundant isotope peak forthe wildtype allele; application of this (external) calibration to thefive other spectra resulted in better than 20 ppm mass accuracy foreach. Differentiation by mass alone of the alleles is straightforward,even for heterozygote mixtures whose components differ by 16.00 (FIG. 67d), 2501 (FIG. 67 e), or 9.01 Da (FIG. 65 f). The value of highperformance MS is clear when recognition of small DNA mass shifts is thebasis for diagnosis of the presence or absence of a mutation. The recentreintroduction of delayed extraction (DE) techniques has improved theperformance of MALDI-TOF with shorts DNAs (Roskey, M. T. et al., (1996)Anal. Chem. 68: 941-946); a resolving power (RP) of >10³ has beenreported for a mixed-base 50-mer, and a pair of 31-mere with a C or a T(Δm 15 Da) at a variable position resolved nearly to baseline. ThusDE-TOF-MS has demonstrated the RP required for separation of theindividual components of heterozygotes. Even with DE, however, theprecision of DNA mass measurement with TOF is typically 0.1% (8 Da at 8kDa) using external calibration, sufficiently high to result inincorrect diagnoses. Despite the possibility of space charge inducedfrequency shifts (Marshall, A. G. et al. (1991) Anal. Chem.63:215A-229A), MALDI-FTMS mass errors are rarely as high as 0.005% (0.4Da at 8 KDa), making internal calibration unnecessary.

The methods for DNA point mutation presented here are not onlyapplicable to the analysis of single base mutations, but also to lessdemanding detection of single or multiple base insertions or deletions,and quantification of tandem two, three, or four base repeats. The PROBEreaction yields products amenable to analysis by relatively lowperformance ESI or MALDI instrumentation; direct measurement of shortamplified product masses is an even more direct means of mutationdetection, and will likely become more widespread with the increasinginterest in high performance MS available with FTMS.

EXAMPLE 16 Immobilization of Nucleic Acids on Solid Supports Via anAcid-Labile Covalent Bifunctional Trityl Linker

Aminolinked DNA was prepared and purified according to standard methods.A portion (10 eq) was evaporated to dryness on a speedvac and suspendedin anhydrous DMF/pyridine (9:1; 0.1 ml). To this was added thechlorotrityl chloride resin (1 eq, 1.05 μmol/mg loading) and the mixturewas shaken for 24 hours. The loading was checked by taking a sample ofthe resin, detritylating this using 80% AcOH, and measuring theabsorbance at 260 nm. Loading was ca. 150 pmol/mg resin.

In 80% acetic acid, the half-life of cleavage was found to besubstantially less than 5 minutes—this compares with trityl ether-basedapproaches of half-lives of 105 and 39 minutes for para and metasubstituted bifunctional dimethoxytrityl linkers respectively.Preliminary results have also indicated that the hydroxy picolinic acidmatrix alone is sufficient to cleave the DNA from the chlorotritylresin.

EXAMPLE 17 Immobilization of Nucleic Acids on Solid Supports ViaHydrophobic Trityl Linker

The primer contained a 5′-dimethoxytrityl group attached using routinetrityl-on DNA synthesis.

C18 beads from an oligo purification cartridge (0.2 mg) placed in afilter tip was washed with acetonitrile, then the solution of DNA (50 ngin 25 μl) was flushed through. This was then washed with 5% acetonitrilein ammonium citrate buffer (70 mM, 250 μl). To remove the DNA form theC18, the beads were washed with 40% acetonitrile in water (10 μl) andconcentrated to ca 2 μl on the Speedvac. The sample was then submittedto MALDI.

The results showed that acetonitrile/water at levels of ca.>30% areenough to dissociate the hydrophobic interaction. Since the matrix usedin MALDI contains 50% acetonitrile, the DNA can be released from thesupport and successfully detected using MALDI-TOF MS (with the tritylgroup removed during the MALDI process).

FIG. 69 is a schematic representation of nucleic acid immobilization viahydrophobic trityl linkers.

EXAMPLE 18 Immobilization of Nucleic Acids on Solid Supports ViaStreptavidin-Iminobiotin

Experimental Procedure

2-iminobiotin N-hydroxy-succinimid ester (Sigma) was conjugated to theoligonucleotides with a 3′- or 5-′ amino linker following the conditionssuggested by the manufacturer. The completion of the reaction wasconfirmed by MALDI-TOF MS analysis and the product was purified byreverse phase HPLC.

For each reaction, 0.1 mg of streptavidin-coated magnetic beads(Dynabeads M-280 Streptavidin from Dynal) were incubated with 80 pmol ofthe corresponding oligo in the presence of 1M NaCl and 50 mM ammoniumcarbonate (pH 9.5) at room temperature for one hour. The beads boundwith oligonucleotides were washed twice with 50 mM ammonium carbonate(pH 9.5). Then the beads were incubated in 2 μl of 3-HPA matrix at roomtemperature for 2 min. An aliquot of 0.5 μl of supernatant was appliedto MALDI-TOF. For biotin displacement experiment, 1.6. mol of freebiotin (80-fold excess to the bound oligo) in 1 μl of 50 mM ammoniumcitrate was added to the beads. After a 5 min. incubation at roomtemperature, 1 μl of 3-HPA matrix was added and 0.5 μl of supernatantwas applied to MALDI-TOF MS. To maximize the recovery of the boundiminobiotin oligo, the beads from the above treatment were againincubated with a 2 μl of 3-HPA matrix and 0.5 μl of supernatant wasapplied to MALDI-TOF MS. The matrix alone and free biotin treatmentquantitatively released iminobiotin oligo off the streptavidin beads asshown in FIGS. 70 and 71.

EXAMPLE 19 Mutation Analysis Using Loop Primer Oligo Base ExtensionMaterials and Methods

Genomic DNA. Genomic DNA was obtained from healthy individuals andpatients suffering from sickle cell anemia. The wildtype and mutatedsequences have been evaluated conventionally by standard Sangersequencing.

PCR-Amplification. PCR amplifications of a part of the β-globin wasestablished and optimized to use the reaction product without a furtherpurification step for capturing with streptavidin coated bead. Thetarget amplification for LOOP-PROBE reactions were performed with theloop-cod5 d(GAG TCA GGT GCG CCA TGC CTC AAA CAG ACA CCA TGG CGC, SEQ IDNO: 58) as forward primer and β-11-bio d(TCT CTG TCT CCA CAT GCC CAG,SEQ ID NO: 59) as biotinylated reverse primer. The underlined nucleotidein the loop-cod5 primer is mutated to introduce an invariant CfoIrestriction site into the amplicon and the nucleotides in italics arecomplementary to a part of the amplified product. The total PCR volumewas 50 μl including 200 ng genomic DNA, 1 U Taq-polymerase(Boehringer-Mannheim, Cat#1596594), 1.5 mM MgCl₂, 0.2 mM dNTPs(Boehringer-Mannheim, Ca#1277049), and 10 pmol of each primer. Aspecific fragment of the β-globin gene was amplified using the followingcycling condition: 5 min 94° C. followed by 40 cycles of: 30 sec @ 94°C., 30 sec @ 56° C., 30 sec @ 72° C., and a final extension of 2 min at72° C.

Capturing and denaturation of biotinylated templates. 10 μl paramagneticbeads coated with streptavidin (10 mg/ml; Dynal, Dynabeads M-280streptavidin Cat#112.06) and treated with 5× binding solution (5 MNH₄Cl, 0.3M NH₄OH) were added to 40 μl PCR volume (10 μl of theamplified product was saved for check electrophoresis). After incubationfor 30 min at 37° C. the supernatant was discarded. The capturedtemplates were denatured with 50 μl 100 mM NaOH for 5 min at ambienttemperature, then washed once with 50 μl 50 mM NH₄OH and three timeswith 100 μl 10 mM Tris.Cl, pH 8.0. The single stranded DNA served astemplates for PROBE reactions.

Primer oligo base extension (PROBE) reaction. The PROBE reactions wereperformed using Sequenase 2.0 (USB Cat# E70775Z including buffer) asenzyme and dNTPs and ddNTPs supplied by Boehringer-Mannheim (Cat#1277049and 1008382). The ratio between dNTPs (dCTP, dGTP, dTTP) and ddATP was1:1 and the total used concentration was 50 μM of each nucleotide. Afteraddition of 5 μl 1-fold Sequenase-buffer the beads were incubated for 5min at 65° C. and for 10 min at 37° C. During this time the partiallyself complementary primer annealed with the target site. The enzymaticreaction started after addition of 0.5 μl 100 mM dithiothreitol (DTT),3.5 μl dNTP/ddNTP solution, and 0.5 μl Sequenase (0.8 U) and incubatedat 37° C. for 10 min. Hereafter, the beads were washed once in 1-fold TEbuffer (10 mM Tris, 1 mM EDTA, pH 8.0).

CfoI restriction digest. The restriction enzyme digest was performed ina total volume of 5 μl using 10 U CfoI in 1-fold buffer L purchased fromBoehringer-Mannheim. The incubation time was 20 min at 37° C.

Conditioning of the Diagnostic Products for Mass Spectrometric Analysis

After the restriction digest, the supernatant was precipitated in 45 μlH₂O, 10 μl 3M NH₄-acetate (pH 6.5), 0.5 μl glycogen (10 mg/ml in water,Sigma, Cat# G1765), and 110 μl absolute ethanol for 1 hour at roomtemperature. After centrifugation at 13,000 g for 10 min the pellet waswashed in 70% ethanol and resuspended in 2 μl 18 Mohm/cm H₂O. The beadswere washed in 100 μl 0.7 M NH₄₋citrate followed by 100 μl 0.05 MNH₄₋citrate. The diagnostic products were obtained by heating the beadsin 2 μl 50 mM NH₄OH at 80° C. for 2 min.

Sample Preparation and Analysis on MALDI-TOF Mass Spectrometry.

Same preparation was performed by mixing 0.6 μl of matrix solution (0.7M 3-hydroxypicolinic acid, 0.07 M dibasic ammonium citrate in 1:1H₂O:CH₃CN) with 0.3 μl of either resuspended DNA/glycogen pellet orsupernatant after heating the beads in 50 mM NH₄OH on a sample targetand allowed to air dry. The sample target was automatically introducedin to the source region of an unmodified Perspective Voyager MALDI-TOFoperated in delayed extraction linear mode with 5 and 20 kV on thetarget and conversion dynode, respectively. Theoretical molecular mass(M_(r)(calc)) were calculated from atomic compositions; reportedexperimental (M_(r)(exp)) values are those of the singly-protonatedform.

Results

The LOOP-PROBE has been applied to the detection of the most commonmutation of codon 6 of the human β-globin gene leading to sickle cellanemia. The single steps of the method are schematically presented inFIG. 72. For the analysis of codon 6, a part of the β-globin gene wasamplified by PCR using the biotinylated reverse primer β11 bio and theprimer loop-cod5 which is modified to introduce a CfoI recognition site(FIG. 72 a). The amplified product is 192 bp in length. After PCR theamplification product was bound to streptavidin coated paramagneticparticles as described above. The antisense strand was isolated bydenaturation of the double stranded amplified product (FIG. 72 b). Theintra-molecule annealing of the complementary 3′ end was accomplished bya short heat denaturation step and incubation at 37° C. The 3′ end ofthe antisense strand is now partially double stranded (FIG. 72 c). Foranalyzing the DNA downstream of the self annealed 3′-end of theantisense strand, the primer oligo base extension (PROBE) has beenperformed using ddATP, dCTP, dGTP, dTTP (FIG. 72 d). This generatesdifferent products in length specific for the genotype of the analyzedindividual. Before the determination of the length of these diagnosticproducts, the DNA was incubated with the CfoI restriction endonucleasethat cuts 5′ of the extended product. This step frees the stem loop fromthe template DNA whereas the extended product still keeps attached tothe template. The extended products are then denatured by heating fromthe template stand and analyzed by MALDI-TOF mass spectrometry.

Since the MALDI-TOF analyses were performed with a non-calibratedinstrument, the mass deviation between observed and expected values wasapproximately 0.6% higher than theoretically calculated. Nevertheless,the results obtained were conclusive and reproducible within repeatedexperiments. In all analyzed supernatants after the restriction digestthe stem loop could be detected. Independent of the genotype, the stemloop has had in all analyses molecular masses about 8150 Da (expected8111 Da). An example is shown in FIG. 73 a. The second peak in thisfigure with a mass of 4076 Da is a doubly charged ion of the stem loop.FIG. 73 b to 73 d show the analyses of different genotypes as indicatedin the respective inserts. HbA is the wildtype genotype and HbC and HbSare two different mutations in codon 6 of the β-globin gene which causesickle cell disease. In the wildtype situation a single peak with amolecular mass of 4247 Da and another with 6696 Da are detected (FIG. 73b). The latter corresponds to the biotinylated PCR primer (β-11-bio)unused in the PCR reaction which also has been removed in someexperiments. The former corresponds to the diagnostic product for HbA.The analyses of the two individual DNA molecules with HbS trait as wellas compound heterozygosity (HbS/HbC) for the sickle cell disorder leadalso to unambiguous expected results (FIGS. 73 c and 73 d).

In conclusion, the LOOP-PROBE is a powerful means for detection ofmutations especially predominant disease causing mutations or commonpolymorphisms. The technique eliminates one specific reagent formutation detection and, therefore, simplifies the process and makes itmore amenable to automation. The specific extended product that isanalyzed is cleaved off from the primer and is therefore shortercompared to the conventional method. In addition, the annealingefficiency is higher compared to annealing of an added primer and shouldtherefore generate more product. The process is compatible withmultiplexing and various detection schemes (e.g., single base extension,oligo base extension and sequencing). For example, the extension of theloop-primer can be used for generation of short diagnostic sequencingladders within highly polymorphic regions to perform, for example, HLAtyping or resistance as well as species typing (e.g., Mycobacteriumtuberculosis)).

EXAMPLE 20 T7-RNA Polymerase Dependent Amplification of CKR-5 andDetection by MALDI-MS Materials and Methods

Genomic DNA. Human genomic DNA was obtained from healthy individuals.

PCR-Amplification and Purification. PCR amplification of a part of theCKR-5 gene was accomplished using ckrT7f as sense primer d(ACC TAG CGTTCA GTT CGA CTG AGA TAA TAC GAC TCA CTA TAG CAG CTC TCA TTT TCC ATA C(SEQ ID NO: 60). The underlined sequence corresponds to the sequencehomologous to CKR-5, the bolded sequence corresponds to the T7-RNApolymerase promoter sequence and the italic sequence was chosenrandomly. ckr5r was used as antisense primer d(AAC TAA GCC ATG TGC ACAACA (SEQ ID NO: 61). Purification of the amplified product and removalof unincorporated nucleotides was carried out using the QIAquickpurification kit (Qiagen, cat#28104). In the final PCR volume of 50 μlwere 200 ng genomic DNA, 1 U Taq-polymerase (Boehringer-Mannheim,cat#1596594), 1.5 mM MgCl₂ 0.2 mM dNTPs (Boehringer-Mannheim,cat#1277049), and 10 pmol of each primer. The specific fragment of theCKR-5 gene was amplified using the following cycling conditions: 5 min @94° C. followed by 40 cycles of 45 sec @ 94° C., 45 sec 52° C., 5 sec @72° C., and a final extension of 5 min at 72° C.

T7-RNA Polymerase conditions. One third of the purified DNA (about 60ng) was used in the T7-RNA polymerase reaction. (Boehringer-Mannheim,cat#881 767). The reaction was carried out for 2 h at 37° C. accordingto the manufacturer's conditions using the included buffer. The finalreaction volume was 20 μl 0.7 μl RNasin (33 U/μl) had been added. Afterthe extension reaction, the enzyme was inactivated by incubation for 5min at 65° C.

DNA Digestion and Conditioning of the Diagnostic Products for Mass SpecAnalysis.

The template DNA was digested by adding RNase-free DNase I(Boehringer-Mannheim, cat#776 758) to the inactivated T7 mixture andincubation for 20 min at room temperature. Precipitation was carried outby adding 1 μl glycogen (10 mg/ml, Sigma, cat# G1765), 1/10 volume 3MNH₂₋acetate (pH 6.5), and 3 volume absolute ethanol and incubation for 1hour at room temperature. After centrifugation at 13,000 g for 10 min,the pellet was washed in 70% ethanol and resuspended in 3 μl 18 Mohm/cmH₂O. 1 μl was analyzed on an agarose gel.

Sample Preparation and Analysis on MALDI-TOF Mass Spectrometry

Sample preparation was performed by mixing 0.6 μl of matrix solution(0.7 M 3-hydroxypicolinic acid, 0.07 M dibasic ammonium citrate in 1:1H₂O:CH₃CN) with 0.3 μl of resuspended DNA/glycogen on a sample targetand allowed to air dry. The sample target was introduced into the sourceregion of an unmodified Finnigan VISION2000 MALDI-TOF operated inreflectron mode with 5 kV. The theoretical molecular mass was calculatedform atomic composition; reported experimental values are those ofsingly-pronated form.

Results

The chemokine receptor CKR-5 has been identified as a major coreceptorin HIV-1 (see e.g., WO 96/39437 to Human Genome Sciences; Cohen, J. etal. Science 275: 1261). A mutant allele that is characterized by a 32 bpdeletion is found in 16% of the HIV-1 seronegative population whereasthe frequency of this allele is 35% lower in the HIV-1 seropositivepopulation. It is assumed that individuals homozygous for this alleleare resistant to HIV-1. The T7-RNA polymerase dependent amplificationwas applied to identify this specific region of the chemokine receptorCKR-5 (FIG. 74). Human genomic DNA was amplified using conventional PCR.The sense primer has been modified so that it contains a random sequenceof 24 bases that facilitate polymerase binding and the T7-RNA polymerasepromoter sequence (FIG. 75). The putative start of transcription is atthe first base 5′ of the promoter sequence. ckr5r was used as anantisense primer. PCR conditions are outlined above. The amplifiedproduct derived from wildtype alleles is 75 bp in length. Primer andnucleotides were separated from the amplification product using theQiagen QIAquick purification kit. One third of the purified product wasapplied to in vitro transcription with T7-RNA polymerase. To circumventinterference of the template DNA, it was digested by adding RNase-freeDNase I. RNA was precipitated and this step also leaves the degraded DNAin the supernatant. Part of the redissolved RNA was analyzed on anagarose gel and the rest of the sample was prepared for MALDI-TOFanalysis. The expected calculated mass of the product is 24560 Da. Adominant peak, that corresponds to an approximate mass of 25378.5 Da canbe observed. Since the peak is very broad, an accurate determination ofmolecular mass was not possible. The peak does not correspond toresidual DNA template. First, the template DNA is digested, and second,the DNA strands would have a mass of 23036.0 and 23174 Da, respectively.

This example shows that T7 RNA polymerase can effectively amplify targetDNA. The generated RNA can be detected by Mass spectrometry. Inconjunction with modified (e.g., 3′-deoxy)ribonucleotides that arespecifically incorporated by a RNA polymerase but not extended anyfurther, this method can be applied to determine the sequence of atemplate DNA.

EXAMPLE 21 MALDI Mass Spectrometry of RNA Endonuclease Digests Materials

Synthetic RNA (Sample A: 5′-UCCGGUCUGAUGAGUCCGUGAGGAC-3′ (SEQ ID NO:62); sample B: 5′-GUCACUACAGGUGAGCUCCA-3′ (SEQ ID NO: 63); sample C:5′-CCAUGCGAGAGUAAGUAGUA-3′ (SEQ ID NO: 64)) samples were obtained fromDNA technology (Aahus, Denmark) and purified on a denaturingpolyacrylamide gel (Shaler, T. A. et al. (1996) Anal. Chem. 63:5766-579). Rnases T₁ (Eurogentec), U₂ (Calbiochem), A(Boehringer-Mannheim) and PhyM (Pharmacia) were used without additionalpurification. Streptavidin-coated magnetic beads (Dynabeads M-280Streptavidin, Dynal) were supplied as a suspension of 6-7×10⁸ bead/ml(10 mg/ml) dissolved in phosphate-buffered saline (PBS) containing 0.1%BSA and 0.02% NaN₃. 3-Hydroxypicolinic acid (3-HPA) (Aldrich) waspurified by a separate desalting step before use as described in moredetail elsewhere (Little, D. P. et al. (1995) Proc. Natl. Acad. Sci.U.S.A. 92: 2318-2322).

Methods

In vitro transcription reaction. The 5′-biotinylated 49 nt in vitrotranscript (SEQ ID NO: 65):AGGCCUGCGGCAAGACGGAAAGACCAUGGUCCCUNAUCUGCCGCAGGAUC was produced bytranscription of the plasmid pUTMS2 (linearized with the restrictionenzyme BamHI) with T7 RNA polymerase (Promega). For the transcriptionreaction 3 μg template DNA and 50 u T7 RNA polymerase were used in a 50μl volume of 1 ul/μl RNA guard (Rnax inhibitor, Pharmacia), 0.5 mM NTP's1.0 mM 5′-biotin-ApG dinucleotide, 40 mM Tris-HCl (pH 8.0), 6 mM MgCl₂ 2mM spermidine and 10 mM DTT. Incubation was performed at 37° C. for 1hour, then another aliquot of 50 units T7 RNA polymerase was added andincubation was continued for another hour. The mixture was adjusted to2M NH₄₋acetate and the RNA was precipitated by addition of one volume ofethanol and one volume of isopropanol. The precipitated RNA wascollected by centrifugation at 20,000×g for 90 min at 4° C., the pelletwas washed with 70% ethanol, dried and redissolved at 8 M urea. Furtherpurification was achieved by electrophoresis through a denaturingpolyacrylamide gel as described elsewhere (Shaler, T. A. et al. (1996)Anal. Chem. 68: 576-579). The ration of 5′-biotinylated tonon-biotinylated transcripts was about 3:1.

Ribonuclease assay. For partial digestion with selected RNases differentenzyme concentrations ad assay conditions were employed as summarized intable VII. The solvents for each enzyme were selected following thesuppliers' instructions. The concentrations of the synthetic RNA samplesand the in vitro transcript were adjusted to 5-10×10⁻⁶M.

TABLE VII Overview and Assay Conditions of the RNAses ConcentrationIncubation Time [units Rnase/ (max. number Rnase Source μgRNA]Conditions of fragments) References T₁ Aspergillus oryzae 0.2 20 mMTris-HCl,   5 min Donis-Keller, H. et al., (1977) pH5.7, 37° C. Nuc.Acids Res. 4: 2527-2537 U₂ Ustilago 0.01 20 mM DAC, pH 5.0, 15 minDonis-Keller, H. et al., (1977) Sphaerogena 37° C. Nuc. Acids Res. 4:2527-2537 PhyM Physarum 20 20 mM DAC, pH 5.0, 15 min Donis-Keller, H. etal., (1980) polycephalu m 50° C. Nucl. Acids Res. 8: 3133-3142 A bovinepancrease 4 × 10⁻⁹ 10 mM Tris-HCl, pH 30 min Breslow, R. and R. Xu. 7.5,37° C. (1993) Proc. Natl. Acad. Sci. USA 90: 1201-1207 CL₃ chicken liver0.01 10 mM Tris-HCl, pH 30 min Boguski, et al., (1980) J. 6.5, 37° C.Biol. Chem. 255: 2160-2163 cusativin cucumis sativus L. 0.05 ng 10 mMTris-HCl, pH 30 min Rojo, M.A. et al. (1994) 6.5, 37° C. Planta 194:328-338

The reaction was stopped at selected times by mixing 0.6 μl aliquots ofthe assay with 1.5 μl of 3 HPA-solution. The solvent was subsequentlyevaporated in a stream of cold air for the MALDI-MS analysis.

Limited alkaline hydrolysis was performed by mixing equal volumes (2.0μl) of 25% ammonium hydroxide and RNA sample (5-10×10⁻⁶ M) at 60° C. 1μl aliquots were taken out at selected times and dried in a stream ofcold air. For these samples it turned out to be important to first drythe digests in a stream of cold air, before 1.5 μl of the matrixsolution and 0.7 μl of NH₄+ loaded cation exchanged polymer beads wereadded.

The reaction was stopped at selected times by mixing 0.6 μl aliquots ofthe assay with 1.5 μl of 3HPA-solution. The solvent was subsequentlyevaporated in a stream of cold air for the MALDI-MS analysis.

Limited alkaline hydrolysis was performed by mixing equal volumes (2.0μl) of 25% ammonium hydroxide and RNA sample (5-10×10⁻⁶ M) at 60° C. 1μl aliquots were taken out at selected times and dried in a stream ofcold air. For these samples it turned out to be important to first drythe digests in a stream of cold air, before 1.5 μl of the matrixsolution and 0.7 μl if a suspension of NH₄ ⁺ loaded cation exchangepolymer beads were added.

Separation of 5′-biotinylated fragments. Steptavidin-coated magneticbeads were utilized to separate 5′-biotinylated fragments of the invitro transcript after partial RNase degradation. The biotin moiety inthis sample was introduced during the transcription reaction initiatedby the 5′-biotin-pApG-dinucleotide. Prior to use, the beads were washedtwice with 2× binding & washing (b&w) buffer (20 mM Tris-HCl, 2 mM EDTA,2 M NaCl pH 8.2) and resuspended at 10 mg/ml in 2×b&w buffer. Circa 25pmol of the RNA in vitro transcript were digested by RNase U2 using theprotocol described above. The digestion was stopped by adding 3 μl of95% formamide containing 10 mMtrans-1,2-diaminocyclohexane-N,N,N¹,N¹-tetraacetic acid (CDTA) at 90° C.for 5 min, followed by cooling on ice. Subsequently, capture of thebiotinylated fragments was achieved by incubation of 6 μl of the digestwith 6 μl of the bead suspension and 3 μl of b&w buffer at roomtemperature for 15 min. Given the binding capacity of the beads of 200pmol of biotinylated oligonucleotide per mg of beads, as specified bythe manufacturer, the almost 2-times excess of oligonucleotide was usedto assure a full loading of the beads. The supernatant was removed, andthe beads were washed twice with 6 μl of H₂O. The CDTA and 95% formamideat 90° C. for 5 min. After evaporation of the solvent and the formamidethe ≦2.5 pmol of fragments were resuspended in 2 μl H₂O and analyzed byMALDI-MS as described above.

Sample preparation for MALDI-MS. 3-Hydroxypicolinic acid (3-HPA) wasdissolved in ultra pure water to a concentration of ca. 300 mM. Metalcations were exchanged against NH₄ ⁺ as described in detail previously.(Little, D. P. et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92:2318-2322). Aliquots of 0.6 μl of the analyte solution were mixed with1.5 μl 3-HPA on a flat inert metal substrate. Remaining alkali cations,present in the sample solution as well as on the substrate surface, wereremoved by the addition of 0.7 μl of the solution of NH₄ ⁺-loaded cationexchange polymer beads. During solvent evaporation, the beadsaccumulated in the center of the preparation, were not used for theanalysis, and were easily removed with a pipette tip.

Instrument. A prototype of the Vision 2000 (ThermBioanalysis, Hemel,Hempstead, UK) reflectron time of flight mass spectrometer was used forthe mass spectrometry. Ions were generated by irradiation with afrequency-tripled ND:YAG laser (355 nm, 5 ns; Spektrum GmbH, Berlin,Germany) and accelerated to 10 ke V. Delayed ion extraction was used forthe acquisition of the spectra shown, as it was found to substantiallyenhance the signal to noise ratio and/or signal intensity. Theequivalent flight path length of the system is 1.7 m, the base pressureis 10⁴⁻ Pa. Ions were detected with a discrete dynode secondary-electronmultiplier (R2362, Hamamatsu Photonics), equipped with a conversiondynode for effective detection of high mass ions. The total impactenergy of the ions on the conversion dynode was adjusted to valuesranging from 16 to 25 keV, depending on the mass to be detected. Thepreamplified output signal of the SEM was digitized by a LeCroy 9450transient recorder (LeCroy, Chestnut Ridge, N.Y., USA) with a samplingrate of up to 400 MHz. For storage and further evaluation, the data weretransferred to a personal computer equipped with custom-made software(ULISSES). All spectra shown were taken in the positive ion mode.Between 20 and 30 single shot spectra were averaged for each of thespectra shown.

Results

Specificity of Rnases. Combining base-specific RNA cleavage withMALDI-MS requires reaction conditions optimized to retain the activityand specificity of the selected enzymes on the one hand and complyingwith the boundary conditions for MALDI on the other. Incompatibilitymainly results because the alkaline-ion buffers, commonly used in thedescribed reaction, such as Na-phosphate, Na-citrate or Na-acetate aswell as EDTA interfere with the MALDI sample preparation; presumablythey disturb the matrix crystallization and/or analyte incorporation.Tris-HCl or ammonium salt buffers, in contrast, are MALDI compatible(Shaler, T. A. et al. (1996) Anal. Chem. 68: 576-579). Moreover,alkaline salts in the sample lead to the formation of a heterogenousmixture of multiple salts of the analyte, a problem increasing withincreasing number of phosphate groups. Such mixtures result in loss ofmass resolution and accuracy as well as signal-to-noise ratio (Little,D. P. et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92: 2318-2322;Nordhoff, E., Cramer, R. Karas, M., Hillenkamp, F., Kirpekar, F.,Kristiansen, K. and Roepstorff, P. (1993) Nucleic Acids Res., 21,3347-3357). Therefore, RNase digestions were carried out under somewhatmodified conditions compared to the ones described in the literature.They are summarized above in table VII. For Rnase T₁, A, CL₃ adCusativin, Tris-HCl (pH 6-7.5) was used as buffer. 20 mM DAC providesthe pH of 5, recommended for maximum activity of RNases U₂ and PhyM. Theconcentration of 10-20 mM of these compounds were found to not interferesignificantly with the MALDI analysis. To examine the specificity of theselected ribonucleases under these conditions, three synthetic 20-25merRNA molecules with different nucleotide sequences were digested.

The MALDI-MS spectra of FIG. 77 shows five different cleavage patterns(A-E) of a 25 nt RNA obtained after partial digestion with RNases T₁,U₂, PhyM, A, and alkaline hydrolysis. These spectra were taken fromaliquots which were removed from the assay after empirically determinedincubation times, chosen to get an optimum coverage of the sequence. Asthe resulting samples were not fractionated prior to mass spectrometricanalysis, they contain all fragments generated at that time by therespective RNases. In practice, uniformity of the cleavages, can beaffected by a preferential attack on the specific phosphodiester bonds(Donis-Keller, H., Maxam, A. M., and Gilbert, W. (1977) Nucleic AcidsRes., 4, 1957-1978; Donis-Keller, H. (1980) Nucleic Acids Res., 83133-3142). The majority of the expected fragments are indeed observedin the spectra. It is also worth noting that for the reaction protocolsas used, correct assignment of all fragment masses is only possible, ifa 2′, 3′-cyclic phosphate group is assumed. It is well known that suchcyclic phosphates are intermediates in the cleavage reaction and gethydrolyzed in a second, independent and slower reaction step involvingthe enzyme (Richards, F. M., and Wycoff, H. W. in The Enzymes Vol. 4,3rd Ed., (ed. Boyer, P. D.) 746-806 (1971, Academic Press, New York);Heinemann, U and W. Saenger (1985) Pure Appl. Chem. 57, 417-422;Ikehara, M. et al., (1987) Pure Appl. Chem. 59-965-968) Vreslow, R. andXu, R. (1993) Proc. Nal. Acad. Sci. USA, 90, 1201-1207). In a few casesdifferent fragments have equal mass of differ by as little as 1 Dalton.In these cases, mass peaks cannot unambiguously be assigned to one orthe other fragments. Digestion of two additional different 20 nt RNAsamples was, therefore, performed (Hahner, S., Kirpekar, F., Nordoff,E., Kristiansen, K., Roepstorff, P. and Hillenkamp, F. (1996)Proceedings of the 44th ASMS Conference on Mass Spectrometry, Portland,Oreg.) in order to sort out these ambiguities. For all samples tested,the selected ribonucleases appear to cleave exclusively at the specifiednucleotides leading to fragments arising from single as well as multiplecleavages.

In FIG. 77, peaks, indicating fragments containing the original5′-terminus, are marked by arrows. All non marked peaks can be assignedto internal sequences or those with retained 3′-terminus. For a completesequence all possible fragments bearing exclusively either the 5′- orthe 3′-terminus of the original RNA would suffice. In practice, the5′-fragments are better suited for this purpose, because the spectraobtained after incubation of all three synthetic RNA samples contain thenearly complete set of originals of 5′-ions for all different RNases(Hahner, S., Kirpekar, F., Nordoff, E., Kristiansen, K., Roepstorff andHillenkamp, F. (1996) Proceedings of the 44th ASMS Conference on MassSpectrometry, Portland, Oreg.). Internal fragments are somewhat lessabundant and fragments containing the original 3′-terminus appearsuppressed in the spectra. In agreement with observations reported inthe literature (Gupta, R. C. and Randerath, K. (1977) Nucleic AcidsRes., 4, 1957-1978), cleavages close to the 3′-terminus were partiallysuppressed in partial digests of the RNA 25 mer by RNase T₁ and U₂ (evenif they are internal or contain the original 5′-terminus). Fragmentsfrom such cleavages appear as weak and poorly resolved signals in themass spectra.

For larger RNA molecules secondary structure is known to influence theuniformity of the enzymatic cleavages (Donis-Keller, H., Maxam, A. M.and Gilbert, A. (1977) Nucleic Acids Res. 8, 3133-3142). This can, inprinciple be, overcome by altered reaction conditions. In assaysolutions containing 5-7 M urea, the activity of RNases such as T₂, U₂,A, Cl₃, and PhyM is known to be retained (Donis-Keller, H., Maxam, A. M.and Gilbert, W. (1977) Nucleic Acids Res., 4, 2527-2537; Boguski, M. S.,Hieter, P. A., and Levy, C. C. (1980) J. Biol. Chem., 255, 2160-2163;Donis-Keller, H. (1980) Nucleic Acids Res., 8, 3133-3142, while RNA issufficiently denatured. UV-MALDI-analysis with 3-HPA as matrix is notpossible under such high concentrations of urea in the sample. Up to aconcentration of 2 M urea in the reaction buffer, MALDI analysis of thesamples was still possible although significant changes in matrixcrystallization were observed. Spectra of the RNA 20 mer (sample B),digested in the presence of 2 M urea still resembled those obtainedunder conditions listed in Table VIl.

Digestion by RNases which exclusively recognize one nucleobase isdesirable to reduce the complexity of the fragment patterns and therebyfacilitate the mapping of the respective nucleobase. RNases CL₃ andcursavitin are enzymes reported to cleave at cytidylic acid residues.Upon limited RNase CL₃ and cursativin digestion of the RNA-20mer (sampleB) under non-denaturing conditions, fragments corresponding to cleavagesat cytidylic residues were indeed observed (FIG. 78). Similar to thedata reported so far (Boguski, M. S., Hieter, P. A. and Levy, C. C.(1980) J. Biol. Chem., 255, 2160-2163: Rojo, M. A., Arias, F. J.,Iglesias, R., Ferreras, J. M., Munoz, R., Escarmis, C., Soriano, F.,Llopez-Fando, Mendez, E., and Girbes, T. (1994) Planta, 194, 328-338).The degradation pattern in FIG. 78, however, reveals that not everycytidine residue is recognized, especially for neighboring C residues.RNase CL₃ is also reported to be susceptible to the influence ofsecondary structure (Boguski, M. S., Heiter, P. A., and Levy, C. C.(1980) J. Biol. Chem., 255, 2160-2163), but for RNA of the size employedin this study, such an influence should be negligible. Therefore,unrecognized cleavage sites in this case can be attributed to a lack ofspecificity of this enzyme. To confirm these data, a further RNaseCL₃-digestion was performed with the RNA 20mer (sample C). As a resultof the sequence of this analyte, all three linkages containing cytidylicacid were readily hydrolyzed, but additional cleavages at uridylic acidresidues were detected as well. Since altered reaction conditions suchas increased temperature (90° C.), various enzyme to substrate ratios,and addition of 2M urea did not result in a digestion of the expectedspecificity, application of this enzyme to sequencing was not pursuedfurther. Introduction of a new cytidine-specific ribonuclease,cusativin, isolated form dry seeds of Cucumis sativus L. lookedpromising for RNA sequencing (Rojo, M. A., Arias, F. J., Iglesias, R.,Ferreras, J. M., Munoz, R., Escarmis, C., Soriano, F., Llopez-Fando, J.,Mendez, E. and Girbes, T. (1994) Planta, 194, 328-338). As shown in FIG.78, not every cytidine residue was hydrolyzed and additional cleavagesoccurred at uridylic acid residues for the recommended concentration ofthe enzyme. RNases CL₃ and cusativin will, therefore not yield thedesired sequence information for mapping of cytidine residues and theiruse was not further pursued. The distinction of pyrimidine residues canbe achieved, however, by use of RNases with multiple specificities, suchas Physarum polycephalum RNase (cleaves ApN, UpN) and pancreatic RNase A(cleaves UpN, CpN) (see FIG. 77). All 5′-terminus fragments, generatedby the monospecific RNase U₂ and apparent in the spectrum of FIG. 77Cwere also evident in the spectrum of the RNase PhyM digest (FIG. 77D).Five of the six uridilic cleavage sites could, this way, be uniquelyidentified by this indirect method. In a next step, the knowledge of theuridine cleavage sites was used to identify sites of cleavage ofcytidilic acid residues in the spectrum recorded after incubation withRNase A (FIG. 77E), again using exclusively ions containing the original5′-terminus. Two of the four expected cleavage sites were identifiedthis way. A few imitations are apparent from these spectra, if only thefragments containing the original 5′-terminus are used for the sequencedetermination. The first two nucleotides usually escape the analysis,because their signals get lost in the low mass matrix background.Because of this, the corresponding fragments are missing in the spectraof the U- and C-specific cleavages. Large fragments with cleavage sitesclose to the 3′-terminus are often difficult to identify, particularlyin digests with RNases T¹, and U₂, because of their low yield (videsupra) and the often strong nearby signal of the non-digestedtranscript. Accordingly the cleavages in position 22 and 23 do not showup in the spectrum of the G-specific RNase T, (FIG. 77A) and thecleavage site 24 cannot be identified from the spectra of the U₂ andPhyM digests (FIGS. 77 C and D). Also site 16 and 17 with twoneighboring cytidilic acids cannot be identified in the RNase A spectrumof FIG. 77E. These observations demonstrate that a determination ofexclusively the 5′-terminus fragments may not always suffice and theinformation contained in the internal fragments may be needed for a fullsequence analysis.

Finally, limited alkaline hydrolysis provides a continuum of fragments(FIG. 77B), which can be used to complete the sequence data. Again, thespectrum is dominated by ions of fragments containing the 5′-terminus,although the hydrolysis should be equal for all phosphodiester bonds. Aswas true for the enzymatic digests, correct mass assignments requiresone to assume that all fragments have a 2′, 3′-cyclic phosphate. Thedistribution of peaks, therefore, resembles that obtained after a3′-exonuclease digest (Pieles, U., Zurcher, W., Schar, M. and Moser, H.E., (1993) Nucleic Acids Res., 21, 3191-3196; Nordhoff, E. et al. (1993)Book of Abstracts, 13^(th) Internat. Mass Spectrom. Conf., Budapest p.218; Kirpekar, F., Nordhoff, E., Kristiansen, K., Roepstorff, P.,Lezius, A. Hahner, S., Karas, M. and Hillenkamp. F. (1994) Nucleic AcidRes., 22, 3866-3870). In principle, the alkaline hydrolysis alone could,therefore, be used for a complete sequencing. This is, however, onlypossible for quite small oligoribonucleotides, because larger fragmentions, differing in mass by only a few mass units will not be resolved inthe spectra and the mass of larger ions cannot be determined with thenecessary accuracy of better than 1 Da, even if peaks are partially orfully resolved. The interpretation of the spectra particularly fromdigests of unknown RNA samples is substantially simplified, if only thefragments containing the original 5′-terminus are separated out prior tothe mass spectrometric analysis. A procedure for this approach isdescribed in the following section.

Separation of 5′-biotinylated fragments. Streptavidin-coated magneticbeads (Dynal) were tested for the extraction of fragments containing theoriginal 5′-terminus from the digests. Major features to be checked forthis solid-phase approach are the selective immobilization and efficientelution of biotinylated species. In preliminary experiments, a5′-biotinylated DNA (19 nt) and streptavidin were incubated and MALDIanalyzed after standard preparation. Despite the high affinity of thestreptavidin-biotin interaction, the intact complex was not found in theMALDI spectra. Instead, signals of the monomeric subunit of streptavidinand the biotinylated DNA were detected. Whether the complex dissociatesin the acidic matrix solution (pKA 3) or during the MALDI desorptionprocess, is not known. Surprisingly, if the streptavidin is immobilizedon a solid surface such as magnetic beads, the same results are notobserved. A mixture of two 5′-biotinylated DNA samples (19 nt and 27 nt)and two unlabeled DNA sequences (12 nt and 22 nt) were incubated withthe beads. The beads were extracted and carefully washed beforeincubation in the 3-HPA MALDI matrix. No analyte signals could beobtained from these samples. To test whether the biotinylated specieshad been bound to the beads altogether, elution form the extracted andwashed beads was performed by heating at 90° C. in the presence of 95%formamide. This procedure is expected to denature the streptavidin,thereby breaking the streptavidin/biotin complex. FIG. 79B shows theexpected signals of the two biotinylated species, proving that releaseof the bound molecules in the MALDI process is the problem rather thanthe binding of the beads; FIG. 79A shows a spectrum of the same sampleafter standard preparation, showing signals of all four analytes as areference. Complete removal of the formamide after the elution and priorto the mass spectrometric analysis was found to be important, otherwisecrystallization of the matrix is disturbed. Mass resolution and thesignal-to-noise ration in spectrum 79B are comparable to those of thereference spectrum. These results testify to the specificity of thestreptavidin-biotin interaction, since no or only minor signals of thenon-biotinylated analyte were detected after incubation with the Dynalbeads. Increased suppression of nonspecific binding was reported throughan addition of the detergent Tween-20 to the binding buffer (Tong, X.and Smith, L. M. (1992) Anal Chem., 64, 2672-2677). Although this effectcould be confirmed in this study, peak broadening affected the qualityof the spectra due to remaining amounts of the detergent. The necessityof an elution step as a prerequisite for detection of the capturedbiotinylated species can be attributed to a stabilizing effect of thecomplex by the immobilization of the streptavidin to the magnetic beads.

For practical application of this solid phase method to sequencing amaximum efficiency of binding and elution of biotinylated species is ofprime importance. Among a variety of conditions investigated so far,addition of salts such as EDTA gave best results in the case of DNAsequencing by providing ionic strength to the buffer (Tong, X. andSmith, L. M. (1992) Anal Chem., 64, 2672-2677). To examine such aneffect on the solid-phase method, several salt additives were tested forthe binding and elution of the 5′-biotinylated RNA in vitro transcript(49 nt). The results are shown in FIG. 80. Judging from the relativeintensity, signal-to-noise ration, and resolution of the respectivesignals, a 95% formamide solution containing 10 mM CDTA (FIG. 80D) ismost efficient for the binding/elution. Since CDTA acts as a chelatingagent for divalent cation, formation of proper secondary an tertiarystructure of the RNA is prevented. An improved sensitivity and spectralresolution has been demonstrated under such conditions for the analysisof RNA samples by electrospray mass spectrometry (Limbach, P. A., Crain,P. F. and McCloskey, J. A. (1995) J Am. Soc. Mass. Spectrom., 6, 27-39).The improvement in the MALDI analysis is actually not very significantcompared to the spectrum obtained for the solution containing formamidealone (FIG. 81 b), but the reproducibility for spectra of good qualitywas substantially improved for the CDTA/formamide solution. Thus inaddition to the improved binding/elution, this additive may also improvethe incorporation of the analyte into the matrix crystals.Unfortunately, a striking signal broadening on the high mass side wasobserved in case of formamide solutions containing EDTA, CDTA or 25%ammonium hydroxide. Since this effect is most prominent in case of 25%ammonium hydroxide and this agent was also used for adjusting EDTA andCDTA to their optimum pH, a pronounced NH₃ adduct ion formation can beassumed.

The applicability of streptavidin-coated magnetic beads separation toRNA sequencing was demonstrated for the Rnase U₂ digest of the5′-biotinylated RNA in vitro transcript (49 nt) (FIG. 81). The entirefragment pattern obtained after incubation with Rnase U₂ is shown isspectrum 81A. Separation of the biotinylated fragments reduces thecomplexity of the spectrum (FIG. 81B) since only 5′-terminal fragmentsare captured by the beads. The signals in the spectrum are broadened andthe increased number of signals in the low mass range indicate that evenafter stringent washing of the beads, some amounts of buffer anddetergent used for the binding and elution remained. Furtherimprovements of the method are, therefore, needed. Another possiblestrategy for application of the magnetic beads is the immobilization ofthe target RNA prior to RNase digestion by an elution of the remainingfragments for further analysis. Cleavage of the RNA was impeded in thiscase, as evidenced by a prolonged reaction time for the digest underotherwise identical reaction conditions.

EXAMPLE 22 Parallel DNA Sequencing Mutation Analysis and MicrosatelliteAnalysis Using Primers with Tags and Mass Spectrometric Detection

This EXAMPLE describes specific capturing of DNA products generated inDNA analysis. The capturing is mediated by a specific tag (5 to 8nucleotides long) at the 5′ end of the analysis product that binds to acomplementary sequence. The capture sequence can be provided by apartially double stranded oligonucleotide bound to a solid support.Different DNA analysis (e.g., sequencing, mutation, diagnostic,microsatellite analysis) can be carried out in parallel, using, forexample, a conventional tube or microtiter plate (MTP). The products arethen specifically captured and sorted out via the complementaryidentification sequence on the tag oligonucleotide. The captureoligonucleotide can be bound onto a solid support (e.g., silicon chip)by a chemical or biological bond. Identification of the sample isprovided by the predefined position of the capture oligonucleotide.Purification, conditioning and analysis by mass spectrometry are done onsolid support. This method was applied for capturing specific primersthat had a 6 base tag sequence.

Materials and Methods

Genomic DNA.

Genomic DNA was obtained from healthy individuals.

PCR Amplification

PCR amplifications of part of the β-globin gene were established usingβ2 d(CATTTGCTTCTGACACAACT SEQ ID NO: 66) as forward primer and β11d(TCTCTGTCTCCACATGCCCAG SEQ ID NO: 67) as reverse primer. The total PCRvolume was 50 μl including 200 ng genomic DNA, 1 U Taq-polymerase(Boehringer-Mannheim, Cat#159594), 1.5 mM MgCl₂, 0.2 mM dNTPs(Boehringer-Mannheim, Cat#1277049), and 10 pmol of each primer. Aspecific fragment of the β-globin gene was amplified using the followingcycling conditions: 5 min @ 94° C. followed by 40 cycles of 30 sec @ 94°C., 45 sec @ 53° C., 30 sec @ 72° C., and a final extension of 2 min @72° C. Purification of the amplified product and removal ofunincorporated nucleotides was carried out using the QIAquickpurification kit (Qiagen, Cat 28104). One fifth of the purified productwas used for the primer oligo base extension (PROBE) or sequencingreactions, respectively.

Primer Oligo Base Extension (PROBE) and Sequencing Reactions

Detection of putative mutations in the human β-globin gene at codon 5and 6 and at codon 30 and in the IVS-1 donor site, respectively, wasdone in parallel (FIG. 82A). β-TAG1 (GTCGTCCCATGGTGCACCTGACTC SEQ ID NO:68) served as primer to analyze codon 5 and 6 and β-TAG2(CGCTGTGGTGAGGCCCTGGGCA SEQ ID NO: 69) for the analyses of codon 30 andthe IVS-1 donor site. The primer oligo base extension (PROBE) reactionwas done by cycling, using the following conditions: final reactionvolume was 20 μl, β-TAG1 primer (5 pmol), β-TAG2 primer (5 pmol), dCTP,dGTP, dTTP, (final concentration each 25 μM), ddATP (final concentration100 μM) dNTPs and ddNTPs purchased from Boeringer-Mannheim, Cat#1277049and 1008382), 2 μl of 10× ThermoSequence buffer and 2.5 UThermoSequenase (Amersham, CAT#E79000Y). The cycling program was asfollows: 5 min @ 94° C., 30 sec @ 53° C., 30 sec @ 72° C. and a finalextension step for 8 min @ 72° C. Sequencing was performed under thesame conditions except that the reaction volume was 25 μl and theconcentration of nucleotides was 250 μM for ddNTP.

Capturing Using TAG Sequence and Sample Preparation

The capture oligonucleotides cap-tag1 d(GACGACGACTGCTACCTGACTCCA SEQ IDNO: 70) and cap-tag2 d(ACAGCGGACTGCTACCTGACTCCA SEQ ID NO: 71),respectively, were annealed to equimolar amounts of uni-asd(TGGAGTCAGGTAGCAGTC SEQ ID NO: 72) (FIG. 82A). Each oligonucleotide hada concentration of 10 pmol/μl in ddH₂O and incubated for 2 min @ 80° C.and 5 min @ 37° C. This solution was stored at −20° C. and aliquots weretaken. 10 pmol annealed capture oligonucleotides were bound to 10 μlparamagnetic beads coated with streptavidin (10 mg/ml; Dynal, DynabeadsM-280 streptavidin Cat# 112.06) by incubation for 30 min @ 37° C. Beadswere captured and the PROBE or sequencing reaction, respectively, wasadded to the capture oligonucleotides. To facilitate binding of β-TAG1and β-TAG2, respectively, the reaction was incubated for 5 min @ 25° C.and for 30 min @ 16° C. The beads were washed twice with ice cold 0.7 MNH₄ Citrate to wash away unspecific bound extension products andprimers. The bound products were dissolved by adding 1 μl DDH₂O andincubation for 2 min @ 65° C. and cooling on ice. 0.3 μl of the samplewere mixed with 0.3 μl matrix solution (saturated 3-hydroxy-picolinicacid, 10% molar ratio ammonium-citrate in acetonitrile/water (50/50.v/v)) and allowed to air dry. The sample target was automaticallyintroduced into the source region of an unmodified Perspective VoyagerMALDI-TOF operated in delayed extraction linear mode with 5 and 20 kV onthe target and conversion dynode, respectively. Theoretical averagemolecular mass (M_(r)(calc)) were calculated from atomic compositions;reported experimental M_(r)(M_(r)(exp)) values are those of thesingly-pronated form.

Results

Specific capturing of a mixture of extension products by a shortcomplementary sequence has been applied to isolate sequencing and primeroligo base extension (PROBE) products. This method was used for thedetection of putative mutations in the human β-globin gene at codon 5and 6 and at codon 30 and IVS-1 donor site, respectively (FIG. 82A).Genomic DNA has been amplified using the primers β2 and β11. Theamplification product was purified and the nucleotides separated. Onefifth of the purified product was used for analyses by primer oligo baseextension. To analyze both sites in a single reaction, primers, β-TAG1and β-TAG2, were used respectively. β-TAG1 binds upstream of codons 5and 6 and β-TAG2 upstream of codon 30 and the IVS-1 donor site.Extension of these primers was performed by cycling in the presence ofddATP and dCTP, dGTP and dTTP, leading to specific products, dependingon the phenotype of the individual. The reactions were then mixed withthe capture oligonucleotides. Capture oligonucleotides include thebiotinylated capture primer cap-tag1 and cap-tag2, respectively. Theyhave 6 bases at the 5′ end, that are complementary to the 5′ end ofβ-TAG1 and β-TAG2, respectively. Therefore, they specifically capturethese primers and the extended products. By annealing a universaloligonucleotide (uni-as) to the capture oligonucleotide, the captureprimer is transformed into a partially double stranded molecule whereonly the capture sequence stays single stranded (FIG. 82). This moleculeis then bound to streptavidin coated paramagnetic particles, to whichthe PROBE or sequencing reaction, respectively is added. The mixture waswashed to bind only the specifically annealed oligonucleotides. Capturedoligonucleotides are dissolved and analyzed by mass spectrometry.

PROBE products of one individual (FIG. 83) show a small peak with amolecular mass of 7282.8 Da. This corresponds to the unextended β-TAG1that has a calculated mass of 7287.8 Da. The peak at 8498.6 Dacorresponds to a product, that has been extended by 4 bases. Thiscorresponds to the wildtype situation. The calculated mass of thisproduct is 8500.6 Da. There is no significant peak indicating aheterozygote situation. Furthermore only β-TAG1 and not β-TAG2 has beencaptured, indicating a high specificity of this method.

Analyses of what was bound to cap-tag2 (FIG. 84) shows only onepredominant peak with a molecular mass of 9331.5 Da. This corresponds toan extension of 8 nucleotides. It indicates a homozygous wildtypesituation where the calculated mass of the expected product is 9355 Da.There is no significant amount of unextended primer and only β-TAG2 hasbeen captured.

To prove that this approach is also suitable for capturing specificsequencing products, the same two primers β-TAG1 and β-TAG2,respectively, were used. The primers were mixed, used in one sequencingreaction and then sorted by applying the above explained method. Twodifferent termination reactions using ddATP and ddCTP were performedwith these primers (FIGS. 85 and 86, respectively). All observed peaksin the spectrograms correspond to the calculated masses in a wildtypesituation.

As shown above, parallel analysis of different mutations (e.g.,different PROBE primers) is now possible. Further, the described methodis suitable for capturing specific sequencing products. Capturing can beused for separation of different sequencing primers out of one reactiontube/well, isolation of specific multiplex-amplified products, PROBEproducts, etc. Conventional methods, like cycle sequencing, andconventional volumes can be used. A universal chip design permits theuse of many different applications. Further, this method can beautomated for high throughput.

EXAMPLE 23 Deletion Detection by Mass-Spectrometry

Various formats can be employed for mass spectrometer detection of adeletion within a gene. For example, molecular mass of a double standardamplified product can be determined, or either or both of the strands ofa double stranded product can be isolated and the mass measured asdescribed in previous examples.

Alternatively, as described herein, a specific enzymatic reaction can beperformed and the mass of the corresponding product can be determined bymass spectrometry. The deletion size can be up to several tenths ofvases in length, still allowing the simultaneous detection of thewildtype and mutated allele. By simultaneous detection of the specificproducts, it is possible to identify in a single reaction whether theindividual is homozygous or heterozygous for a specific allele ormutation.

Materials and Methods

Genomic DNA

Leukocyte genomic DNA was obtained from unrelated healthy individuals.

PCR Amplification

PCR amplification of the target DNA was established and optimized to usethe reaction products without a further purification step for capturingwith streptavidin coated beads. The primers for target amplification andfor PROBE reactions were as follows:

CKRΔ-F: d(CAG CTC TCA TTT TCC ATA C SEQ ID NO: 73) and CKRΔ-R bio: d(AGCCCC AAG ATG ACT ATC SEQ ID NO: 74). CKR-5 was amplified by the followingprogram: 2 min @ 94° C., 45 seconds @ 52° C., 5 seconds @ 72° C., and afinal extension of 5 minutes at 72° C. The final volume was 50 μlincluding 200 ng genomic DNA 1 U Taq-polymerase (Boehringer-Mannheim,Cat #1596594), 1.5 Mm MgCl₂, 0.2 Mm DNTPS (Boehringer-Mannheim, Cat#1277049), 10 pmol of unmodified forward primers, and 8 pmol 5′biotinylated reverse primer.

Capturing and Denaturation of Biotinylated Templates

10 μl paramagnetic beads coated with streptavidin (10 mg/ml; Dynal,Dynabeads M-280 streptavidin Cat #112.06) in 5× binding solution (5MNH₄Cl, 0.3 M NH₄OH) were added to 45 μl PCR reaction (5 μl of PCRreaction were saved for electrophoresis). After binding by incubationfor 30 min. at 37° C. the supernatant was discarded. Captured templateswere denatured with 50 μl of 100 Mm NaOH for 5 min. at ambienttemperature, washed once with 50 μl 50 Mm NH₄OH and three times with 100μl 10 Mm Tris/Cl, Ph 8.0. The single stranded DNA served as templatesfor PROBE reactions.

Primer Oligo Base Extension (PROBE) Reaction

The PROBE reaction was performed using Sequence 2.0 (USB Cat # E70775Zincluding buffer). dATP/DGTP and ddTTP were supplied byBoehringer-Mannheim (Cat #1277049 and 1008382). d(CAG CTC TCA TTT TCCATA C (SEQ ID NO: 73) was used as PROBE primer (FIG. 87). The followingsolutions were added to the beads: 3.0 μl H₂O, 1.0 μl reaction buffer,1.0 μl PROBE primer (10 pmol) and incubated at 65° C. for 5 minutesfollowed by 37° C. for 10 min. Then 0.5 μl DTT, 3.5 μl DNTPS/ddntp each50 μM and 0.5 μl Sequenase (0.8 U) were added and incubated at 37° C.for 10 min.

T4 Treatment of DNA

To generate blunt ended DNA, amplification products were treated with T4DNA polymerase (Boehringer-Mannheim Cat#1004786). The reactions werecarried out according to the manufacturer's protocol for 20 min. at 11°C.

Direct Size Determination of Extended Products

To determine the size of the amplified product, MALDI-TOF was applied toone strand of the amplification product. samples were bound to beads, asdescribed above, conditioned and denatured, as described below.

DNA Conditioning

After the PROBE reaction the supernatant was discarded and the beadswere washed first in 50 μl 700 mM NH₄-citrate and second 50 μl 50 mMNH₄-citrate. The generated diagnostic products were removed for thetemplate by heating the beads in 2 μl H₂O at 80° C. for 2 min. Thesupernatant was used for MALDI-TOF analysis.

Sample Preparation and Analysis with MALDI-TOF Mass

Spectrometry

Sample preparation was performed by mixing 0.6 μl of matrix solution(0.7 M 3-hydroxypicolinic acid, 0.07 M dibasic citrate in 1:1 H₂O:CH₃CN)with 0.3 μl of diagnostic PROBE products in water on a sample target andallowed to air dry. Up to 100 samples were spotted on a probe targetdisk for introduction into the source region of an unmodifiedPerspective Voyager MALDI-TOF instrument operated in linear mode withdelayed extraction and 5 and 30 kV on the target and conversion dynode,respectively. Theoretical average molecular mass (M_(r)(calc)) ofanalytes were calculated from atomic compositions, reported experimentalM_(r)(M_(r)(exp)) values are those of the singly-pronated form,determined using internal calibration with unextended primers in thecase of PROBE reactions.

Conventional Analyses

Conventional analyses were performed by native polyacrylamide gelelectrophoresis according to standard protocols. The diagnostic productswere denatured with formamide prior to loading onto the gels and stainedwith ethidium bromide or silver, respectively.

Results

The CKR-5 status of 10 randomly chosen DNA samples of healthyindividuals were analyzed. Leukocyte DNA was amplified by PCR and analiquot of the amplified product was analyzed by standard polyacrylamidegel electrophoresis and silver staining of the DNA (FIG. 88). Foursamples showed two bands presumably indicating heterozygosity for CKR-5,whereas the other 6 samples showed one band, corresponding to ahomozygous gene (FIG. 88). In the case where two bands were observed,they correspond to the expected size of 75 bp for the wildtype gene and43 bp for the allele with the deletion (FIG. 87). Where one band wasobserved, the size was about 75 bp which indicated a homozygous wildtypeCKR-5 allele. One DNA sample derived from a presumably heterozygous onefrom a homozygous individual were used for all further analysis. Todetermine the molecular mass of the amplified product, DNA was subjectedto matrix assisted laser desorption/ionization coupled with time offlight analysis (MALDI-TOF). Double stranded DNA, bound to streptavidincoated paramagnetic particles, was denatured and the strand releasedinto the supernatant was analyzed. FIG. 89A shows a spectrograph of aDNA sample, that was supposed to be heterozygous according to the resultderived by polyacrylamide gel electrophoresis (FIG. 88). The calculatedmass of the sense strand for a wildtype gene is 23036 Da and for thesense strand carrying the deletion allele 13143 (FIG. 87 and Table VI).Since many thermostable polymerases unspecifically add an adenosine tothe 3′ end of the product, those masses were also calculated. They are23349 and 13456 Da. The masses of the observed peaks (FIG. 89A) are23119 Da, which corresponds to the calculated mass of a wildtype DNAstrand where an adenosine has been added (23349 Da). Since no peak witha mass of about 23036 Da was observed, the polymerase must havequalitatively added adenosine. Two peaks, which are close to each other,have a mass of 13451 and 13137 Da. This corresponds to the calculatedmasses of the allele, with the 32 bp deletion. The higher mass peakcorresponds to the product, where adenosine has been added and the lowermass peak to the one without the unspecific adenosine. Both peaks haveabout the same height, indicating that to about half of the productadenosine has been added. The peak with a mass of 11682 Da is a doublycharged molecule of the DNA corresponding to 23319 Da (2×11682 Da=23364Da). The peaks with masses of 6732 and 6575 Da are doubly chargedmolecules of the one with masses of 13451 and 13137 Da and the peak with7794 Da corresponds to the triply charged molecule of 23319Da. Multiplecharged molecules are routinely identified by calculation. Amplified DNAderived from a homozygous individual shows in the spectrograph (FIG.89C) one peak with a mass 23349.6 and a much smaller peak with a mass of23039.9 Da. The higher mass peak corresponds to DNA resulting from awildtype allele with an added adenosine, that has a calculated mass of23349 Da. The lower mass peak corresponds to the same product withoutadenosine. Three further peaks with a mass of 11686, 7804.6 and 5852.5Da correspond to doubly, triply and quadruply charged molecules.

The unspecific added adenine can be removed from the amplified DNA bytreatment of the DNA and T4 DNA polymerase. DNA derived from aheterozygous and a homozygous individual was analyzed after T4 DNApolymerase treatment. FIG. 89B shows the spectrograph derived fromheterozygous DNA. The peak corresponding to the wildtype strand has amass of 23008 Da indicating that the added adenine had been removedcompletely. The same is observed for the strand with a mass of 13140 Da.

The other three peaks are multiply charged molecules of the parentpeaks. The mass spectrograph for the homozygous DNA shows one peak thathas a mass of 23004 Da, corresponding to the wildtype DNA strand withoutan extra adenine added. All other peaks are derived from multiplycharged molecules of this DNA. The amplified products can be analyzed bydirect determination of their masses, as described above, or bymeasuring the masses of products, that are derived from the amplifiedproduct in a further reaction. In this “primer oligo base extension(PROBE)” reaction, a primer that can be internal, as it is in the nestedPCR, or identical to one of the PCR primers, is extended for just a fewbases before the termination nucleotide is incorporated. Depending onthe extension length, the genotype can be specified. CKRΔ-F was used asa PROBE primer, and dATP/dGTP and ddTTP as nucleotides. The primerextension is AGT in case of a wildtype template and AT in case of thedeletion (FIG. 87). The corresponding masses are 6604 Da for thewildtype and 6275 Da for the deletion, respectively. PROBE was appliedto two standard DNAs. The spectrograph (FIG. 90A) shows peaks withmasses of 6604 Da corresponding to the wildtype DNA and at 6275 Dacorresponding to the CKR-5 deletion allele (Table VIII). The peak at amass of 5673 Da corresponds to CKRΔ-F (calculated mass of 5674 Da).Further samples were analyzed in analogous way (FIG. 90B). It isunambiguously identified as homozygous DNA, since the peak with a massof 6607 Da corresponds to the wildtype allele and the peak with a massof 5677 Da to the unextended primer. No further peaks were observed.

The example demonstrates that deletion analysis can be performed by massspectrometry. As shown herein, the deletion can be analyzed by directdetection of single stranded amplified products, or by analysis ofspecifically generated diagnostic products (PROBE). In addition, asshown in the following Example 26, double stranded DNA amplifiedproducts can be analyzed.

Size Calculated Mass Measured Mass wildtype w/o A 2303623039/23009/23004 wildtype with A 23349 23319/23350 deletion w/o A 1314313137/13139 deletion with A 13456 13451 PROBE wildtype 6604  6604/6608deletion 6275  6275All masses are in Dalton.

EXAMPLE 24 Pentaplex tc-PROBE Summary

The multiplexing of thermocycling primer oligo base extension (tc-PROBE)was performed using five polymorphic sites in three differentapolipoprotein genes, which are thought to be involved in thepathogenesis of atherosclerosis. The apolipoprotein A IV gene (codons347 and 360), the apolipoprotein E gene (codons 112 and 158), and theapolipoprotein B gene (codon 3500) were examined. All mass spectra wereeasy to interpret with respect to the five polymorphic sites.

Materials and Methods

PCR Amplification

Human leukocytic genomic DNA was used for PCR. Listed below are theprimers used for the separated amplification of portions of the Apo AIV, Apo E and the Apo B genes:

Apo A IV: A347F: 5′-CGA GGA GCT CAA GGC GAG AAT-3′ (SEQ ID NO: 75) A360R-2-bio: *5′-CAG GGG CAG CTC AGC TCT C-3′ (SEQ ID NO: 76) Apo E: ApoE-F:5′-GGC ACG GCT GTC CAA GGA-3′ (SEQ ID NO: 77) ApoE-R bio: *5′-AGG CCGCGC TCG GCG CCC TC-3′ (SEQ ID NO: 78) Apo B: ApoB-F2 bio: *5′-CTT ACTTGA ATT CCA AGA GC-3′ (SEQ ID NO: 79) Apo B-R: 5′-GGG CTG ACT TGC ATGGAC CGG A-3′ (SEQ ID NO: 80) *biotinylated

Taq polymerase and 10× buffer were purchased from Boehringer-Mannheim(Germany) and dNTPs for Pharmacia (Freiburg, Germany). The total PCRreaction volume was 50 μl including 10 pmol of each primer and 10% DMSO(dimethylsulfoxide, Sigma) (no DMSO for the PCR of the Apo B gene), with˜200 mg of genomic DNA used as template and a final dNTP concentrationof 200 μM. Solutions were heated to 80° C. before the addition of 1 UTaq polymerase; PCR conditions were: 5 min at 95° C., followed by 2cycles 30 sec 94° C., 30 sec 62° C., 30 sec 72° C., 2 cycles 30 sec 94°C. 30 sec 58° C., 30 sec 72° C., 35 cycles of 30 sec at 94° C., 30 secat 56° C., 30 sec at 72° C., and a final extension time of 2 min at 72°C. To remove unincorporated primers and nucleotides, amplified productswere purified using the “QIAquick” (Qiagen, Germany) kit, with elutionof the purified products in 50 μL of TE buffer (10 mM Tris-HCl, 1 mMEDTA, pH 8.0).

Binding of the Amplified Product on Beads

10 μl of each purified amplified product was bound to 5 μl DynaBeads(Dynal, M-280 Streptavidin) and denatured according to the protocol fromDynal. For the pentaplex tc-PROBE reaction the three different amplifiedproduct (bound on the beads) were pooled.

Tc-PROBE

For the PROBE reaction the following primers were used:

(Apo A) P347: (SEQ ID NO: 81) 5′-AGC CAG GAC AAG-3′ (Apo A) P360: (SEQID NO: 82) 5′-ACA GCA GGA ACA GCA-3′ (Apo E) P112: (SEQ ID NO: 83)5′-GCG GAC ATG GAG GAC GTG-3′ (Apo E) P158: (SEQ ID NO: 84) 5′-GAT GCCGAT GAC CTG CAG AAG-3′ (Apo B) P3500: (SEQ ID NO: 85) 5′-GTG CCC TGC AGCTTC ACT GAA GAC-3′

The tc-PROBE was carried out in a final volume of 25 μl containing 10pmol of each primer listed above, 2.5 U Thermoquenase (Amersham), 2.5 μLThermoquenase buffer, and 50 μM dTTP (final concentrations) and 200 μMof ddA/C/GTP, respectively. Tubes containing the mixture were placed ina thermocycler and subjected to the following cycling conditions:denaturation (94° C.) the supernatant was carefully removed from thebeads and ‘desalted’ by ethanol precipitation to exchange nonvolatilecations such as Na+ and K+ with NH₄+, which evaporated during theionization process; 5 μL 3M ammonium acetate (pH 6.5) 0.5 μL glycogen(10 mg/mL, Sigma), 25 μL H₂O, and 110 μL absolute ethanol were added to25 μL PROBE supernatant and incubated for 1 hour at 4° C. After a 10min. centrifugation at 13,000×g, the pellet was washed in 70% ethanoland resuspended in 1 μL 18 Mohm/cm H₂O. A 0.35 μL aliquot of resuspendedDNA was mixed with 0.35 μL matrix solution (0.7 M 3-hydroxypicolinicacid (3-HPA), 0.07 M ammonium citrate in 1:1 H₂0:CH₃CN) on a stainlesssteel sample target disk and allowed to air dry preceding spectrumacquisition using the Thermo Bioanalysis Version 2000 MALDI-TOF operatedin reflectron mode with 5 and 20 kV on the target and conversion dynode,respectively, Theoretical average molecular masses (M₁(calc)) of thefragments were calculated from atomic compositions. External calibrationgenerated from synthetic (ATCG)_(n) oligonucleotide (3.6-18 kDa) wasused. Positive ion spectra from 1-37500 Da were collected.

Results

Table VIII shows the calculated molecular masses of all possibleextension products including the mass of the primer itself. FIG. 91shows a respective MALDI-TOP MS spectra of a tc-PROBE using threedifferent templates and 5 different PROBE primers simultaneously in onereaction. Comparison of the observed and calculated masses (see tableVIII) allows a fast genetic profiling of various polymorphic sites in anindividual DNA sample. The sample presented in FIG. 91 is homozygous forthreonine and glutamine at position 347 and 360, respectively, in theapolipoprotein A IV gene, bears the epsilon 3 allele homozygous in theapolipoprotein E gene, and is also homozygous at the codon 3500 forarginine in the apolipoprotein B gene.

TABLE VIII SEQ ID NO: mass allele Apolipoprotein A IV5′-AGCCAGGACAAG-3′ (347) 86 3688.40 unextended primer5′-AGCCAGGACAAGTC-3 87 4265.80 347 Ser 5-AGCCAGGACAAGA-3′ 88 3985.60 347Thr 5′-ACAGCACCAACAGCA-3′(360) 89 4604.00 unextended primer5′-ACAGCAGGAACAGCATC-3′ 90 5181.40 360 His 5′-ACAGCAGGAACAGCAG-3′ (112)91 4917.20 360 Gln Apolipoprotein E 5′-GCGGACATGGAGGACGTG-3′ (112) 925629.60 unextended primer 5′-GCGGACATGGAGGACGTGGC-3′ 93 6247.00 112 Cys5′-GCGGACATGGAGGACGTGC-3′ 94 5902.80 112 Arg5′-GATGCCGATGACCTGCAGAAG-3′ (158) 95 6480.20 unextended primer5′-GATGCCGATGACCTGCAGAAGC-3′ 96 6753.40 158 Arg5′-GATGCCGATGACCTGCAGAAGTG-3′ 97 7097.60 158 Cys Apolipoprotein B-1005′-GTGCCCTGCAGCTTCACTGAAGAC-3′ 98 7313.80 unextended (3500) primer5′-GTGCCCTGCAGCTTCACTGAAGACTG-3′ 99 7931.20 3500 Gln5′-GTGCCCTGCAGCTTCACTGAAGACC-3′ 100 7587.00 3500 Arg

EXAMPLE 25 Sequencing Exons 5 to 8 of the p53 Gene by MALDI-TOF MassSpectrometry Materials & Methods

Thirty-five cycles of PCR reactions were performed in a 96 wellmicroliter plate with each well containing a total volume of 50 μlincluding 200 ng genomic DNA, 1 unit Taq DNA polymerase, 1.5 mM Mg C1₂,0.2 mM dNTPx, 10 pmol of the forward primer and 6 or 8 of thebiotinylated reverse primer. The sequences of PCR primers preparedaccording to established chemistry (N. D. Sinha, J. Biernat, H. Kter,Tetrahed. Lett. 24: 5843-5846 (1983) are as follows: exon 5:d(biotin-

exon 5: d(biotin-TATCTGTTCACTTGTGCCC SEQ ID NO: 101) andd(biotin-CAGAGGCCTGGGGACCCTG SEQ ID NO: 102); exon 6:D(ACGACAGGGCTGGTTGCC SEQ ID NO: 103) and d(biotin-ACTGACAACCACCCTTAACSEQ ID NO: 104); exon 7: d(CTGCTTGCCACAGGTCTC SEQ ID NO: 105) andd(biotin-CACAGCAGGCCAGTGTGC SEQ ID NO: 106; exon 8:d(GGACCTGATTTCCTTACTG SEQ ID NO: 107) and d(biotin-TGAATCTGAGGCATAACTGSEQ ID NO: 108).

To each well of the 96-well microliter plate containing unpurifiedamplified product, 0.1 mg of paramagnetic streptavidin beads (Dynal) in10 μl of 5× binding solution (5 M NH₄OH) was added and incubated at 3 7°C. for 30 min.

Then beads were treated with 0.1 M NaOH at room temperature for 5 minfollowed by one wash with 50 mM NH₄OH at room temperature for 5 minfollowed by one wash with 50 mM Tris-HCl.

Four dideoxy termination reactions were carried out in separate wells ofthe microliter plate. A total of 84 reactions (21 primers×4reactions/primer) can be performed in a single microliter plate. To eachwell containing immobilized single-stranded template, a total volute of10 μl reaction mixture was added including 1× reaction buffer, 10 pmolof sequencing primer, 250 mM of dNTPs, 25 mM of one of the ddNTPs, and1˜2 units of Thermosequenase (Amersham). Sequencing reactions werecarried out on a thermal cycler using non-cycling conditions: 80° C., 1min, 50° C., 1 min, 50° C. to 72° C., ramping 0.1°/sec, and 72° C., 5min. The beads were then washed with 0.7 M ammonium citrate followed by0.05 M ammonium citrate. Sequencing products were then removed frombeads by heating the beads to 80° C. in 2 μl of 50 mM NH₄OH for 2 min.The supernatant was used for MALDI-TOF MS analysis.

Matrix was prepared as described in Kter, et al (Kter, H. et al., NatureBiotechnol. 14: 1123-1128 (1996)). This saturated matrix solution wasthen diluted 1.52 times with pure water before use. 0.3 μl of thediluted matrix solution was then diluted 1.52 times with pure waterbefore use. 0.3 μl of the diluted matrix solution was loaded onto thesample target and allowed to crystallize followed by addition of 0.3 μlof the aqueous analyte. A Perspective Voyager DE mass spectrometer wasused for the experiments, and the samples were typically analyzed in themanual mode. The target and middle plate were kept at +18.2 kV for 200nanoseconds after each laser shot and then the garget voltage was raisedto +20 kV. the ion guide wire in the flight tube was kept at −2V.Normally, 250 laser shots were accumulated for each sample. ̂Theoriginal spectrum was acquired under 500 MHz digitizing rate, and thefinal spectrum was smoothed by a 455 point average (Savitsky and Golay,(1964) Analytical Chemistry; 36:1627). Default calibration of the massspectrometer was used to identify each peak and assign sequences. Thetheoretical mass values of two sequencing peaks were used to recalibrateeach spectrum. (D. P. Little, T. J. Cornish, M. J. O'Donnel, A. Braun,R. J. Cotter, H. Kter, Anal. Chem., submitted).

Results

Alterations of the p53 gene are considered to be a critical step in thedevelopment of many human cancers (Greenblatt, et al., (1994) CancerRes. 54, 4855-4878; C. C. Harris, (1996) J. Cancer, 73, 261-269; and D.Sidransky and M. Hollstein, (1996) Annu. Res. Med., 47, 285-301).Mutations may serve as molecular indicators of clonality or as earlymarkers of relapse in a patient with a previously identified mutation ina primary tumor (Hainaut, et al., (1997) Nucleic Acid Res., 25,151-157). The prognosis of the cancer may differ according to the natureof the p53 mutations present (H. S. Goh et al., (1995) Cancer Res, 55,5217-5221). Since the discovery of the p53 gene, more than 6000different mutations have been detected. Exons 5-8 were selected assequencing targets where most of the mutations cluster (Hainaut et al.(1997) Nucleic Acids Res., 25, 151-7).

FIG. 96 schematically depicts the single tube process for targetamplification and sequencing, which was performed, as described indetail in the Materials and Methods. Each of exon 5-8 of the p53 genewas PCR amplified using flanking primers in the intron region; the downstream primer was biotinylated. Amplifications of different exons wereoptimized to use the same cycling profile, and the products were usedwithout further purification. PCR reactions were performed in a 96 wellmicroliter plate and the product generated in one well was used as thetemplate for one sequencing reaction. Streptavidin-coated magnetic beadswere added to the same microliter plate and amplified products wereimmobilized. The beads were then treated with NaOH to generateimmobilized single-stranded DNA as sequencing template. The beads werewashed extensively with Tris buffer since remaining base would reducethe activity of sequencing enzyme.

A total of 21 primers were selected to sequence exon 5-8 of the p53 geneby primer walking. The 3′-end nucleotide of all the primers is locatedat the site where no known mutation exists. Four termination reactionswere performed separately which resulted in a total of 84 sequencingreactions on the same PCR microliter plate. Non-cycling conditions wereadopted for sequencing since streptavidin coated beads do not toleratethe repeated application of high temperature. Sequencing reactions weredesigned so that mt terminated fragments were under 70 nucleotides, asize range easily accessible by MALDI-TOF MS and yet long enough tosequence through the next primer binding site. Thermequenase was theenzyme of choice since it could reproducible generate a high yield ofsequencing products in the desired mass range. After the sequencingreactions, the beads were washed with ammonium ion buffers to replaceall other cations. The sequencing ladders were then removed from thebeads by heating in ammonium hydroxide solution or simply in water.

A sub-microliter aliquot of each of the 84 sequencing reactions wasloaded onto one MS sample holder containing preloaded matrix. FIG. 94gives an example of sequencing data generated from one primer; fourspectra are superimposed.

All sequencing peaks were well resolved in the mass range needed to readthrough the next sequencing primer site. Sometimes doubly charged peakswere observed which could be easily identified by correlating the massto that of the singly charged ion. False stops generated by earlytermination of the enzymatic extension can be observed close to theprimer site. Since the mass resolution is high enough, it is easy todifferentiate the false stop peaks from the real sequencing peaks bycalculating the mass difference of the neighboring peaks and crscomparing the four spectra. Additionally, mt primers generateddetectable data through the region of the downstream primer binding sitethereby covering the false stop region.

Using optimized procedures of amplification, sequencing, andconditioning, exons 5-8 of the p53 gene were successfully sequenced.Correct wildtype sequence data were obtained from all exons with a massresolution about 300 to 800 over the entire mass range. The overall massaccuracy is 0.05% or better. The average amount of each sequencingfragment loaded on the MS sample holder is estimated to be 50 fmol orless.

This example demonstrates the feasibility of sequencing exons of a humangene by MALDI-TOF MS. Compare to gel-based automated fluorescent DNAsequencing, the read lengths are shorter. Microchip technology can beincorporated to provide for parallel processing. Sequencing productsgenerated in the microtiter plate can be directly transferred to amicrochip which serves as a launching pad for MALDI-TOF MS analysis.Robot-driven serial and parallel nanoliter dispensing tools are beingused to produce 100-1000 element DNA arrays on <1″ square chips withflat or geometrically altered (e.g., with wells) surfaces for rapid massspectrometric analysis.

FIG. 94 shows an MS spectrum obtained on a chip where the sample wastransferred from a microtiter plate by a pintool. The estimated amountof each termination product loaded is 5 fmol or less which is in therange of amounts used in conventional Sanger sequencing withradiolabeled or fluorescent detection (0.5-1 fmol per fragment). The lowvolume MALDI sample deposition has the advantages of miniaturization(reduced reagent cts), enhanced reproducibility and automated signalacquisition.

EXAMPLE 26 Direct Detection of Synthetic and Biologically GeneratedDouble-Stranded DNA by MALDI-TOF MS Introduction

Typically, matrix-associated laser desorption/ionization (Karas, et.al., (1989) Int. J. Mass Spectrom, Ion Processes, 92, 231)time-of-flight mass spectrometry (MALDI-TOF MS) of DNA molecules whichare double stranded (ds) in solution yields molecular ionsrepresentative of the two single stranded components (Tang, et al.(1994) Rapid Commun. Mass Spectrom. 8: 183; Tang, et al. (1995) NucleicAcids Res. 23: 3126; Benner, et al. (1995) Rapid Commun. Mass Spectrom.9: 537; Liu, et al. (1995) Anal. Chem. 67: 3482; Siegert et al. (1996)Anal. Biochem. 243: 55; and Doktycz, et al. (1995) Anal. Biochem. 230:205); this has been observed in several reports dealing withbiologically generated DNA from a polymerase chain reaction (PCR)amplification (Tang, et al. (1994) Rapid Commun. Mass Spectrom. 8: 183;Liu, et al. (1995) Anal. Chem. 67: 3482; Siegert et al. (1996) Anal.Biochem. 243: 55; and Doktycz, et al. (1995) Anal. Biochem. 230: 205).It is not clear whether the double strand is destabilized because of thedecreased pH in the matrix environment or because of absorbance by theduplex during desorption/ionization/acceleration of an energy sufficientto overcome the attractive van der Waals and “stacking” stabilizationforces (Cantor and Shimmel, Biophysical Chemistry Part I: Theconformation of Biomolecules, W.H. Freeman, New York, (1980), 176). Whenanalyte is present at high concentrations formation of non-specificgas-phase DNA multimers is, as with proteins (Karas, et. al., (1989)Int. J. Mass Spectrom, Ion Processes 92:231), common; however, Lecchiand Pannell (Lecchi et al. (1995) J. Am. Soc. Mass Spectrom. 6: 972)have provided strong evidence for specific Watson Crick (WC) basepairing being maintained in the gas phase. They detected these specificdimers when using 6-aza-2-thiothymine as a matrix, but did not observethem with 3-hydroxypicolinic acid (3-HPA) or 2,4,6-hydroxyacetophenonematrix. As described below, by using a low acceleration voltage of theions and preparing samples for MALDI analysis at reduced temperatures,routine detection of dsDNA is possible.

Materials and Methods

Synthetic DNA. Oligonucleotides were synthesized (Sinha, et al. (1984)Nucleic Acids Res., 12, 4539) on a Perspective Expedite DNA synthesizerand reverse phase HPLC purified in-house. Sequences were: 50-mer (15337Da): 5′-TTG CGT ACA CAC TGG CCG TCG TTT TAC AAC GTC GTG ACT GGG AAA ACCCT-3′ (SEQ ID NO: 109); 27-mer_(c) (complementary, 8343 Da): 5′-GTA AAACGA CGG CCA GTG TGT ACG CAA-3′ (SEQ ID NO: 110); 27-mer_(nc)(non-complementary, 8293 Da): 5′-TAC TGG AAG GCG ATC TCA GCA ATC AGC-3′(SEQ ID NO: 111). 100 μM stock solutions were diluted to 20, 10, 5, and2.5 μM using 18 Mohm/cm H₂O. 2 μL each of equimolar solutions of the50-mer and either 27-mer_(c) or 27-mer_(nc) were mixed and allowed toanneal at room temperature for 10 minutes. 0.5 μL of these mixtures weremixed directly on a sample target with 1 μL matrix (0.7 M 3-HPA, 0.07 Mammonium citrate in 50% acetonitrile) and allowed to air dry.

Biological DNA. Enzymatic digestion of human genomic DNA from leukocyteswas performed. PCR primers (forward, 5′-GGC ACG GCT GTC CAA GGA G-3′(SEQ ID NO: 112)); reverse, 5′-AGG CCG CGC TCG GCG CCC TC-3′ (SEQ ID NO:113) to amplify a portion of exon 4 of the apolipoprotein E gene weredelineated from the published sequence (Das et al., (1985) J. Biol.Chem., 260 6240). Taq polymerase and 10× buffer were purchased fromBoehringer-Mannheim (Germany) and dNTPs from Pharmacia (Freiburg,Germany). The total reaction volume was 50 μl including 20 pmol of eachprimer and 10% DMSO (dimethylsulfoxide, Sigma) with approximately 200 ngof genomic DNA used as template. Solutions were heated to 80° C. beforethe addition of IU polymerase; PCR conditions were: 2 min at 94° C.,followed by 40 cycles of 30 sec at 94° C., 45 sec at 63° C., 30 sec at72° C., and a final extension time of 2 min at 72° C. While noquantitative data was collected to determine the final yield ofamplified product, it is estimated that −2 pmol were available for theenzymatic digestion.

CfoI and RsaI and reaction buffer L were purchased fromBoehringer-Mannheim. 20 μl of amplified products were diluted with 15 μlwater and 4 μl buffer L; after addition of 10 units of restrictionenzymes the samples were incubated for 60 min at 37° C. Forprecipitation of digest products 5 μl of 3M ammonium acetate (pH 6.5),(5 μl glycogen (Braun, et al. (1997) Clin. Chem. 43: 1151) (10 mg/ml,Sigma), and 110 μl absolute ethanol were added to 50 μL of the analytesolutions and stored for 1 hour at room temperature. After at 10 mincentrifugation at 13,000×g, the pellet was washed in 70% ethanol andresuspended in 1 μl 18 Mohm/cm H₂O.

Sample preparation and analysis by MALDI-TOF MS. 0.35 μl of resuspendedDNA was mixed with 0.35-1.3 μL matrix solution (0.7M 3-hydroxypicolinicacid (3-HPA), 0.07 M ammonium citrate in 1:1H₂O:CH₃CN) (Wu, et al.(1993) Rapid Commun. Mass Spectrom. 7: 142) on a stainless steel sampletarget disk and allowed to air dry preceding spectrum acquisition usinga Thermo Bioanalysis Vision 2000 MALDI-TOF instrument operated inpositive ion reflectron mode with 5 and 20 kV on the target andconversion dynode, respectively. Theoretical average molecular masses(M_(r)(calc)) of the fragments were calculated from atomic compositions;the mass of a proton (1.08 Da) was subtracted from raw data values inreporting experimental molecular masses (M_(r)(exp)) as neutral basis.External calibration generated from eight peaks (2000-18000 Da) was usedfor all spectra.

Results and Discussion

FIG. 96A is a MALDI-TOF mass spectrum of a mixture of the synthetic50-mer with (non-complementary) 27-mer_(nc) (each 10 μM, the highestfinal concentration used in this study); the laser power was adjusted tojust above the threshold irradiation for ionization. The peaks at 8.30and 15.34 kDa represent singly charged ions derived from the 27- and50-mer single strands, respectively. Poorly resolved low intensitysignals at −16.6 and −30.7 kDa represent homodimers of 27- and 50-mer,respectively; that at 23.6 kDa is consistent with a heterodimercontaining one 27-mer and one 50-mer strand. Thus low intensity dimerions representing all possible combinations from the twonon-complementary oligonucleotides (27+27; 27+50; 50+50) were observed.Increasing the irradiance even to a point where depurination peaksdominated the spectrum resulted in slightly higher intensities of thesedimer peaks. Note that the hybridization was performed at roomtemperature and with a very low salt concentration, conditions at whichnon-specific hybridization may occur.

FIG. 96 shows a MALDI-TOF spectrum of the same 50-mer mixed with(complementary) 27-mer_(c); the final concentration of eacholigonucleotide was again 10 μM. Using the same laser power as in FIG.96A, intense signals were again observed at 88.34 and 15.34 Kda,consistent with single stranded 27- and 50-mer, respectively. Homodimerpeaks (27+27; 50+50) were barely apparent in the noise; however, singly(23.68 Kda) and doubly (11.84 k Da) charged heterodimer (27+50) peakswere dominant. Although the 23.68 Kda dimer peak could be detected fromall irradiated positions, its intensity relative to the monomer peaksvaried slightly from spot-to-spot. Repeating the experiment withindividual oligonucleotide concentrations of 5, 2.5, and 1.25 μMresulted in decreasing amounts of the 27-/50-mer Watson-Crick dimer peakrelative to the 27- and 50-mer single stranded peaks. At the lowestconcentrations, the observation of dimer was “crystal-dependent”, thatis, irradiation of some crystals produced significant 27-/50-mer dimersignal, while other crystals reproducibly yielded very little or none.This indicates that the incorporation of dsdna into the matrix crystalsor the effectiveness of retaining this interaction through theionization/desorption process is dependent upon the microscopicproperties of the crystals, and/or that there exist steep concentrationgradients of the duplex throughout the sample.

Thus the FIG. 96 spectra provide strong evidence that specific WC basepaired dsdna can be observed using gentle laser conditions with highconcentrations of oligonucleotides in this mass range, the first reportof this using a 3-HPA matrix. The study was extended to a complexmixture of dsdna derived from an enzymatic digest (RsaI/CfoI) of aregion of exon 4 of the apolipoprotein E gene (Das et. al., (1985) J.Biol. Chem., 260 6240); expected fragment masses are given in Table IX.

TABLE IX CfoI/RsaI Digestion Products from ApoE gene exon 4² bases^(b)ssDNA (Da) (+) (−) (+) (−) dsdna (Da) 11 13 3428 4025 7453 16 5004 49249928 18 5412 5750 11162 17 19 5283 5880 11163 19 5999 5781 11780 24 227510 6745 14225 31 29 9628 9185 18813 36 38 11279 11627 22906 48 1484514858 29703 55 53 17175 16240 33415 ^(a)ε3 allele has no 17/19 or 19/19pairs; ε4 allele contains no 36/38 pair. ^(b)(+) sense strand, (−)antisense strandAfter the digestion step, the samples were purified and concentrated byethanol precipitation and resuspended in 1 μL H₂O before mixing them atroom temperature with matrix on the sample target. Nearly 20 peaksranging in mass from 3.4-17.2 Kda were resolved in the products' MALDIspectrum (FIG. 97A), all consistent with denatured single strandedcomponents of the double strand (Table IX). Many such analyses ofsimilar biological products over a period of months also yielded spectrawith negligible dsdna, consistent with previous reports (Tang, et al.(1994) Rapid Commun. Mass Spectrom. 8: 183; Liu, et al. (1995) Anal.Chem. 67: 3482; Siegert et al. (1996) Anal. Biochem. 243: 55; andDoktycz, et al. (1995) Anal. Biochem. 230: 205); contrarily, intactdouble strands were observed under similar conditions for the syntheticDNA (FIG. 96A). It is difficult to estimate the strand concentrationavailable after the biological reactions, but presumably that it was farlower than that at which dimerization of synthetic samples occurred.Furthermore, maintaining specific hybrids within the two-componentsynthetic mixture may be kinetically favored relative to the far morecomplex mixture of 20 single-stranded DNA components from the digest.

The effect of reduced temperature on maintaining dsDNA was tested. Analiquot of the digested DNA solution, the matrix, pipette, pipette tips,and the stainless steel sample target were stored in a 4° C. “cold room”for 15 minutes; as with normal preparations matrix, and then analyte,were spotted on the target and allowed to co-crystallize while airdrying. Crystallization for mixtures of 300 nL 3HPA (50% acetonitrile)with 300 nL analyte required ˜1 minute at room temperature but ˜15minutes at the reduced temperature. Sample spots prepared in the coldroom environment typically contained a high proportion of largetransparent crystals.

MALDI-TOF analysis of an ApoE digest aliquot prepared at reducedtemperature produced the FIG. 97B spectrum. While the low mass rangeappeared qualitatively similar to FIG. 97A, dramatic differences above 8kDa were observed. Only signals consistent with single strands (TableIX) were observed in FIG. 97A, but the FIG. 97B cold room preparedsamples did not yield signals for the same masses except below 8 kDa.Even more striking were the additional high mass peaks in FIG. 97B;clearly these represent dimer peaks containing lower mass components. Aswas done with the synthetic DNA, it was important to determine whetherthese represent non-specific heterodimers, specific WC heterodimers, ornonspecific homodimers. Consider first the 33.35 kDa fragment. Ignoringthe unlikely possibility that the high mass fragment represents a trimeror higher multimer, as a dimer it must only contain the highest massssDNA components, i.e., the >16 kDa. Homodimerization of the 15.24 and17.18 kDa fragments would result in 32.49 and 34.35 kDa peaks,respectively; corresponding mass errors for these incorrect assignmentsrelative to the observed 33.35 kDa would be −2.6% and +3.0%respectively. A far better match is achieved if this peak originatesfrom a heterodimer of the two highest mass single stranded fragments;their summed mass (16.24+17.18=33.42 kDa) differed by 0.2% from theobserved dimer mass 33.35 kDa, an acceptable mass error for MALDI-TOFanalysis of large DNA fragments using external calibration. Likewise,the 29.66 kDa fragment was measured only 0.13% lower than the 29.70 Daexpected for a heterodimer of 48-mers; the sum of no other possiblehomodimers or heterodimers were within a reasonable range of this mass.Similar arguments could be made for the 22.89 and 18.83 kDa fragments,representing 36-/38-mer and 31-/29-heterodimers, respectively; thesignal at 14.86 kDa is consistent with singly charged single strandedand doubly charged double-stranded 48-mer. The agreement of the FIG. 97Bmasses above 15 kDa with the of dsDNA expected from this digest and theabsence of homodimers and non-specific heterodimers at random massesindicated that the base pairings were indeed highly specific andprovided further evidence that gas-phase WC interactions may be retainedin MALDI-generated ions.

FIG. 98 shows a MALDI-TOF spectrum of an ε4 allele, which, unlike theε3, was expected to yield no 36-/38-mer pair upon CfoI/RsaI digestion.The ε3 and ε4 mass spectra were similar except that abundant 22.89 kDafragment in FIG. 97B was not present in FIG. 98; with this informationalone (Table IX) ε3 and ε4 alleles were easily distinguished, therebydemonstrating the genotyping by direct measurement of dsDNA by MALDI-TOFMS. Similarly dsDNA could be ionized, transferred to the gas phase, anddetected by MALDI-TOF MS. The acceleration voltage typically employed onour instrument was only −5 kV corresponding to 1.5 kV/mm up to −2 mmfrom the sample target, with the electric field strength decreasingrapidly with distance from the sample target. Most previous work used atleast 20 kV acceleration (Lecchi et al. (1995) J. Am. Soc. MassSpectrom. 6: 972); in one exception a 27-mer dsDNA was detected using afrozen matrix solution and 100 V acceleration (Nelson, et al. (1990)Rapid Commun. Mass Spectrom. 4: 348). Without being bound by any theoryMALDI-induced “denaturation” of dsDNA may be due to gas-phasecollisional activation that disrupts the WC pairing when highacceleration fields are employed, analogous to the denaturation presumedto be a first step in the fragmentation used for sequencing the singlestranded components of dsDNA using electrospray ionization (McLaffertyet al. (1996) Int. J. Mass Spectrom., Ion Processes). It appears thatthe high salt concentrations (typically >10 mM NaCl or KCl) required tostabilize WC paired dsDNA in solution are unsuitable for MALDI analysis(Nordhoff et al. (1993) Nucleic Acids Res. 21: 3347); reducing theconcentration of such non-volatile cations is necessary to avoidcation-adducted MALDI signals, but destabilizes the double strands insolution. The low pH conditions of the matrix environment should alsodestabilize the duplex. As shown in FIGS. 97B and 98, storing andpreparing even low concentrations of the biological samples at reducedtemperature at least in part offset these denaturing effects, especiallyfor longer strands where melting temperatures are higher due to a moreextensive hydrogen bonding network. The conditions used here arerecognized to be very non-stringent annealing conditions.

The low mass tails on high mass dsDNA peaks (e.g., FIG. 97B, 232 kDa)are consistent with depurination generated to a higher extent than thesum of depurination from each of the single strands combined. Althoughdepurination in solution is an acid-catalyzed reaction, the weaklyacidic conditions in the 3-HPA matrix do not induce significantdepurination; molecular ion signals from a mixed-base 50-mer measuredwith De-MALDI-TOF had only minor contributions from depurination peaks(Juhaz, et al. (1996) Anal. Chem. 68: 941). Depurination from the singlestranded components of the gas-phase dsDNA is observed even though thesebases are expected to be hydrogen bonded to the complementary base ofthe accompanying strand, implying that covalent bonds are being brokenbefore the strand is denatured.

EXAMPLE 27 Efficiency and Specificity Assay for Base-SpecificRibonucleases

Aliquots sampled at regular time intervals during digestion of selectedsynthetic 20 to 25 mers were analyzed by mass spectrometry. Three of theRNAses were found to be efficient and specific. These include: theG-specific T₁, the A-specific U₂ and the A/U-specific PhyM. Theribonucleases presumed to be C-specific were found to be less reliable,e.g., did not cleave at every C or also cleaved at U in an unpredictablemanner. The three promising RNAses all yielded cleavage at all of thepredicted positions and a complete sequence coverage was obtained. Inaddition, the presence of cleavage products containing one or severaluncleaved positions (short incubation times), allowed alignment of thecleavage products. An example of the MALDI-spectrum of an aliquotsampled after T₁ digest of a synthetic 20-mer (SEQ ID NO: 114) RNA isshown in FIG. 100.

EXAMPLE 28 Immobilization of Amplified DNA Targets to Silicon Wafers

Silicon Surface Preparation

Silicon wafers were washed with ethanol, flamed over bunsen burner, andimmersed in an anhydrous solution of 25% (by volume)3-aminopropyltriethoxysilane in toluene for 3 hours. The silane solutionwas then removed, and the wafers were washed three times with tolueneand three times with dimethyl sulfoxide (DMSO). The wafers were thenincubated in a 10 mM anhydrous solution of N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) (Pierce Chemical, Rockford, Ill.) inanhydrous DMSO. Following the reaction, the SIAB solution was removed,and the wafers were washed three times with DMSO. In all cases, theiodoacetamido-functionalized wafers were used immediately to minimizehydrolysis of the labile iodoacetamido-functionality. Additionally, allfurther wafer manipulations were performed in the dark since theiodoacetamido-functionality is light sensitive.

Immobilization of Amplified Thiol-Containing Nucleic Acids

The SIAB-conjugated silicon wafers were used to analyze specific freethiol-containing DNA fragments of a particular amplified DNA targetsequence. A 23-mer oligodeoxynucleotide containing a 5′-disulfidelinkage (purchased from Operon Technologies; SEQ ID NO: 117) that iscomplementary to the 3′-region of a 112 bp human genomic DNA templateGenebank Acc. No.: Z52259; SEQ ID NO: 118) was used as a primer inconjunction with a commercially available 49-mer primer, which iscomplementary to a portion of the 5′-end of the genomic DNA (purchasedfrom Operon Technologies; SEQ ID NO: 119), in PCR reactions to amplify a135 bp DNA product containing a 5′-disulfide linkage attached to onlyone strand of the DNA duplex (SEQ ID NO: 120).

The PCR amplification reactions were performed using the AmplitaqGoldKit (Perkin Elmer Catalog No. N808-0249). Briefly, 200 ng 112 bphuman genomic DNA template was incubated with 10 μM of 23-mer primer and8 μM of commercially available 49-mer primer, 10 mM dNTPs, 1 unit ofAmplitaq Gold DNA polymerase in the buffer provided by the manufacturerand PCR was performed in a thermocycler.

The 5′-disulfide bond of the resulting amplified product was fullyreduced using 10 mM tris-(2-carboxyethyl)phosphine (TCEP) (PierceChemical, Rockford, Ill.) to generate a free 5′-thiol group. Disulfidereduction of the modified oligonucleotide was monitored by observing ashift in retention time on reverse-phase FPLC. It was determined thatafter five hours in the presence of 10 mM TCEP, the disulfide was fullyreduced to a free thiol. Immediately following disulfide cleavage, themodified oligonucleotide was incubated with theiodacetamido-functionalized wafers and conjugated to the surface of thesilicon wafer through the SIAB linker. To ensure complete thioldeprotonation, the coupling reaction was performed at pH 8.0. Using 10mM TCEP to cleave the disulfide and the other reaction conditionsdescribed above, it was possible to reproducibly yield a surface densityof 250 fmol per square mm of surface.

Hybridization and MALDI-TOF Mass Spectrometry

The silicon wafer conjugated with the 135 bp thiol-containing DNA wasincubated with a complementary 12-mer oligonucleotide (SEQ ID NO: 121)and specifically hybridized DNA fragments were detected using MALDI-TOFMS analysis. The mass spectrum revealed a signal with an observedexperimental mass-to-charge ratio of 3618.33; the theoreticalmass-to-charge ratio of the 12-mer oligomer sequence is 3622.4 Da.

Thus, specific DNA target molecule that contain a 5′-disulfide linkagecan be amplified. The molecules are immobilized at a high density on aSIAB-derivatized silicon wafer using the methods described herein andspecific complementary oligonucleotides may be hybridized to thesetarget molecules and detected using MALDI-TOF MS analysis.

EXAMPLE 29 Use of High Density Nucleic Acid Immobilization to GenerateNucleic Acid Arrays

Employing the high density attachment procedure described in EXAMPLE 28,an array of DNA oligomers amenable to MALDI-TOF mass spectrometryanalysis was created on a silicon wafer having a plurality of locations,e.g., depressions or patches, on its surface. To generate the array, afree thiol-containing oligonucleotide primer was immobilized only at theselected locations of the wafer (e.g., see EXAMPLE 28). The eachlocation of the array contained one of three different oligomers. Todemonstrate that the different immobilized oligomers could be separatelydetected and distinguished, three distinct oligonucleotides of differinglengths that are complementary to one of the three oligomers werehybridized to the array on the wafer and analyzed by MALDI-TOF massspectrometry.

Oligodeoxynucleotides

Three sets of complementary oligodeoxynucleotide pairs were synthesizedin which one member of the complementary oligonucleotide pair contains a3′- or 5′-disulfide linkage (purchased from Operon Technologies orOligos, Etc.). For example, Oligomer 1 (d(CTGATGCGTCGGATCATCTTTTTT-SS);SEQ ID NO: 122) contains a 3′-disulfide linkage whereas Oligomer 2(d(SS-CCTCTTGGGAACTGTGTAGTATT); a 5′-disulfide derivative of SEQ ID NO:117) and Oligomer 3 (d(SS-GAATTCGAGCTCGGTACCCGG); a 5′-disulfidederivative of SEQ ID NO: 115) each contain a 5′-disulfide linkage.

The oligonucleotides complementary to Oligomers 1-3 were designed to beof different lengths that are easily resolvable from one another duringMALDI-TOF MS analysis. For example, a 23-mer oligonucleotide (SEQ ID NO:123) was synthesized complementary to a portion of Oligomer 1, a 12-meroligonucleotide (SEQ ID NO: 121) was synthesized complementary to aportion of Oligomer 2 and a 21-mer (SEQ ID NO: 116) was synthesizedcomplementary to a portion of Oligomer 3. In addition, a fourth 29-meroligonucleotide (SEQ ID NO: 124) was synthesized that lackscomplementarity to any of the three oligomers. This fourtholigonucleotide was used as a negative control.

Silicon Surface Chemistry and DNA Immobilization

(a) 4×4 (16-Location) Array

A 2×2 cm² silicon wafer having 256 individual depressions or wells inthe form of a 16×16 well array was purchased from a commercial supplier(Accelerator Technology Corp., College Station, Tex.). The wells were800×800 μm², 120 μm deep, on a 1.125 pitch. The silicon wafer wasreacted with 3-aminopropyltriethoxysilane to produce a uniform layer ofprimary amines on the surface and then exposed to the heterobifunctionalcrosslinker SIAB resulting in iodoacetamido functionalities on thesurface (e.g., see EXAMPLE 28).

To prepare the oligomers for coupling to the various locations of thesilicon array, the disulfide bond of each oligomer was fully reducedusing 10 mM TCEP as depicted in EXAMPLE 28, and the DNA resuspended at afinal concentration of 10 μM in a solution of 100 mM phosphate buffer,pH 8.0. Immediately following disulfide bond reduction, the free-thiolgroup of the oligomer was coupled to the iodoacetamido functionality at16 locations on the wafer using the probe coupling conditionsessentially as described above in EXAMPLE 28. To accomplish the separatecoupling at 16 distinct locations of the wafer, the entire surface ofthe wafer was not flushed with an oligonucleotide solution but, instead,an ˜30-nl aliquot of a predetermined modified oligomer was added inparallel to each of 16 locations (i.e., depressions) of the 256 wells onthe wafer to create a 4×4 array of immobilized DNA using a roboticpintool.

The robotic pintool consists of 16 probes housed in a probe block andmounted on an X Y, Z robotic stage. The robotic stage was a gantrysystem which enables the placement of sample trays below the arms of therobot. The gantry unit itself is composed of X and Y arms which move 250and 400 mm, respectively, guided by brushless linear servo motors withpositional feedback provided by linear optical encoders. A lead screwdriven Z axis (50 mm vertical travel) is mounted to the xy axis slide ofthe gantry unit and is controlled by an in-line rotary servo motor withpositional feedback by a motor-mounted rotary optical encoder. The workarea of the system is equipped with a slide-out tooling plate that holdsfive microtiter plates (most often, 2 plates of wash solution and 3plates of sample for a maximum of 1152 different oligonucleotidesolutions) and up to ten 20×20 mm wafers. The wafers are placedprecisely in the plate against two banking pins and held secure byvacuum. The entire system is enclosed in plexi-glass housing for safetyand mounted onto a steel support frame for thermal and vibrationaldamping. Motion control is accomplished by employing a commercial motioncontroller which was a 3-axis servo controller and is integrated to acomputer; programming code for specific applications is written asneeded.

To create the DNA array, a pintool with assemblies that have solid pinelements was dipped into 16 wells of a multi-well DNA source platecontaining solutions of Oligomers 1-3 to wet the distal ends of thepins, the robotic assembly moves the pin assembly to the silicon wafer,and the sample spotted by surface contact. Thus, one of modifiedOligomers 1-3 was covalently immobilized to each of 16 separate wells ofthe 256 wells on the silicon wafer thereby creating a 4×4 array ofimmobilized DNA.

In carrying out the hybridization reaction, the three complementaryoligonucleotides and the negative control oligonucleotide were mixed ata final concentration of 10 μM for each oligonucleotide in 1 ml of TEbuffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) supplemented with 1 M NaCl,and the solution was heated at 65° C. for 10 min. Immediatelythereafter, the entire surface of the silicon wafer was flushed with 800μl of the heated oligonucleotide solution. The complementaryoligonucleotides were annealed to the immobilized oligomers byincubating the silicon array at ambient temperature for 1 hr, followedby incubation at 4° C. for at least 10 min. Alternatively, theoligonucleotide solution can be added to the wafer which is then heatedand allowed to cool for hybridization.

The hybridized array was then washed with a solution of 50 mM ammoniumcitrate buffer for cation exchange to remove sodium and potassium ionson the DNA backbone (Pieles et al., (1993) Nucl. Acids Res. 21:3191-3196). A 6-nl aliquot of a matrix solution of 3-hydroxypicolinicacid (0.7 M 3-hydroxypicolinic acid-10% ammonium citrate in 50%acetonitrile; see Wu et al. Rapid Commun. Mass Spectrom. 7: 142-146(1993)) was added in series to each location of the array using arobotic piezoelectric serial dispenser (i.e., a piezoelectric pipettesystem).

The piezoelectric pipette system is built on a system purchased fromMicrodrop GmbH, Norderstedt Germany and contains a piezoelectric elementdriver which sends a pulsed signal to a piezoelectric element bonded toand surrounding a glass capillary which holds the solution to bedispensed; a pressure transducer to load (by negative pressure) or empty(by positive pressure) the capillary; a robotic xyz stage and robotdriver to maneuver the capillary for loading, unloading, dispensing, andcleaning, a stroboscope and driver pulsed at the frequency of the piezoelement to enable viewing of ‘suspended’ droplet characteristics;separate stages for source and designation plates or sample targets(i.e. Si chip); a camera mounted to the robotic arm to view loading todesignation plate; and a data station which controls the pressure unit,xyz robot, and piezoelectric driver.

The 3-HPA solution was allowed to dry at ambient temperature andthereafter a 6-nl aliquot of water was added to each location using thepiezoelectric pipette to resuspend the dried matrix-DNA complex, suchthat upon drying at ambient temperature the matrix-DNA complex forms auniform crystalline surface on the bottom surface of each location.

MALDI-TOF MS Analysis

The MALDI-TOF MS analysis was performed in series on each of the 16locations of the hybridization array illustrated in FIG. 6 essentiallyas described in EXAMPLE 28. The resulting mass spectrum ofoligonucleotides that specifically hybridized to each of the 16locations of the DNA hybridization revealed a specific signal at eachlocation representative of observed experimental mass-to-charge ratiocorresponding to the specific complementary nucleotide sequence.

For example, in the locations that have only Oligomer 1 conjugatedthereto, the mass spectrum revealed a predominate signal with anobserved experimental mass-to-charge ratio of 7072.4 approximately equalto that of the 23-mer; the theoretical mass-to-charge ratio of the23-mer is 7072.6 Da. Similarly, specific hybridization of the 12-meroligonucleotide to the array, observed experimental mass-to-charge ratioof 3618.33 Da (theoretical 3622.4 Da), was detected only at thoselocations conjugated with Oligomer 2 whereas specific hybridization ofMJM6 (observed experimental mass-to-charge ratio of 6415.4) was detectedonly at those locations of the array conjugated with Oligomer 3(theoretical 6407.2 Da).

None of the locations of the array revealed a signal that corresponds tothe negative control 29-mer oligonucleotide (theoretical mass-to-chargeratio of 8974.8) indicating that specific target DNA molecules can behybridized to oligomers covalently immobilized to specific locations onthe surface of the silicon array and a plurality of hybridization assaysmay be individually monitored using MALDI-TOF MS analysis.

(b) 8×8 (64-Location) Array

A 2×2 cm² silicon wafer having 256 individual depressions or wells thatform a 16×16 array of wells was purchased from a commercial supplier(Accelerator Technology Corp., College Station, Texas). The wells were800×800 μm², 120 μm deep, on a 1.125 pitch. The silicon wafer wasreacted with 3-aminopropyltriethoxysilane to produce a uniform layer ofprimary amines on the surface and then exposed to the heterobifunctionalcrosslinker SIAB resulting in iodoacetamido functionalities on thesurface as described above.

To make an array of 64 elements, a pintool was used following theprocedures described above. The pintool was dipped into 16 wells of a384 well DNA source plate containing solutions of Oligomers 1-3, movedto the silicon wafer, and the sample spotted by surface contact. Next,the tool was dipped in washing solution, then dipped into the same 16wells of the source plate, and spotted onto the target 2.25 mm offsetfrom the initial set of 16 spots; the entire cycle was repeated to makea 2×2 array from each pin to produce an 8×8 array of spots (2×2elements/pin×16 pins=64 total elements spotted).

Oligomers 1-3 immobilized to the 64 locations were hybridized tocomplementary oligonucleotides and analyzed by MALDI-TOF MS analysis. Asobserved for the 16-location array, specific hybridization of thecomplementary oligonucleotide to each of the immobilizedthiol-containing oligomers was observed in each of the locations of theDNA array.

EXAMPLE 30 Extension of Hybridized DNA Primers Bound to DNA TemplatesImmobilized on a Silicon Wafer

The SIAB-derivatized silicon wafers can also be employed for primerextension reactions of the immobilized DNA template using the proceduresessentially described in EXAMPLE 7.

A 27-mer oligonucleotide (SEQ ID NO: 125) containing a 3′-free thiolgroup was coupled to a SIAB-derivatized silicon wafer as describedabove, for example, in EXAMPLE 28. A 12-mer oligonucleotide primer (SEQID NO: 126) was hybridized to the immobilized oligonucleotide and theprimer was extended using a commercially available kit (e.g., Sequenaseor ThermoSequenase, U.S. Biochemical Corp). The addition of SequenaseDNA polymerase or ThermoSequenase DNA polymerase in the presence ofthree deoxyribonucleoside triphosphates (dNTPs; dATP, dGTP, dCTP) anddideoxyribonucleoside thymidine triphosphate (ddTTP) in buffer accordingto the instructions provided by the manufacturer resulted in a 3-baseextension of the 12-mer primer while still bound to the silicon wafer.The wafer was then analyzed by MALDI-TOF mass spectrometry as describedabove. The mass spectrum results clearly distinguish the 15-mer (SEQ IDNO: 127) from the original unextended 12-mer thus indicating thatspecific extension can be performed on the surface of a silicon waferand detected using MALDI-TOF MS analysis.

EXAMPLE 31 Effect of Linker Length on Polymerase Extension of HybridizedDNA Primers Bound to DNA Templates Immobilized on a Silicon Wafer

The effect of the distance between the SIAB-conjugated silicon surfaceand the duplex DNA formed by hybridization of the target DNA to theimmobilized oligomer template was investigated, as well as choice ofenzyme.

Two SIAB-derivatized silicon wafers were conjugated to the 3′-end of twofree thiol-containing oligonucleotides of identical DNA sequence exceptfor a 3-base poly dT spacer sequence incorporated at the 3′-end:

CTGATGCGTC GGATCATCTT TTTT SEQ ID NO: 122 CTGATGCGTC GGATCATCTT TTTTTTT.SEQ ID NO: 125These oligonucleotides were synthesized and each was separatelyimmobilized to the surface of a silicon wafer through the SIABcross-linker (e.g., see EXAMPLE 28). Each wafer was incubated with a12-mer oligonucleotide:

AAAAAAGATG AT SEQ ID NO: 126 GATGATCCGA CG SEQ ID NO: 128 GATCCGACGC AT,SEQ ID NO: 129which is complementary to portions of the nucleotide sequences common toboth of the oligonucleotides, by denaturing at 75° C. and slow coolingthe silicon wafer. The wafers were then analyzed by MALDI-TOF massspectrometry as described above.

As described in EXAMPLE 30 above, a 3-base specific extension of thebound 12-mer oligonucleotide was observed using the oligomer primerwhere there is a 9-base spacer between the duplex and the surface (SEQID NO: 125). Similar results were observed when the DNA spacer lengthsbetween the SIAB moiety and the DNA duplex were 0, 3, 6 and 12. Inaddition, the extension reaction may be performed using a variety of DNApolymerases, such as Sequenase and Thermo Sequenase (US Biochemical).Thus, the SIAB linker may be directly coupled to the DNA template or mayinclude a linker sequence without effecting primer extension of thehybridized DNA.

EXAMPLE 32 Spectrochip Mutant Detection in ApoE Gene

This example describes the hybridization of an immobilized template,primer extension and mass spectrometry for detection of the wildtype andmutant Apolipoprotein E gene for diagnostic purposes. This exampledemonstrates that immobilized DNA molecules containing a specificsequence can be detected and distinguished using primer extension ofunlabeled allele specific primers and analysis of the extension productsusing mass spectrometry.

A 50 base synthetic DNA template complementary to the coding sequence ofallele 3 of the wildtype apolipoprotein E gene:

(SEQ ID NO: 280)5′-GCCTGGTACACTGCCAGGCGCTTCTGCAGGTCATCGGCATCGCGGAGGAG-3′or complement to the mutant apolipoprotein E gene carrying a G→Atransition at codon 158:

(SEQ ID NO: 281)5′-GCCTGGTACACTGCCAGGCACTTCTGCAGGTCATCGGCATCGCGGAGGAG-3′containing a 3′-free thiol group was coupled to separateSIAB-derivatized silicon wafers as described in Example 28.

A 21-mer oligonucleotide primer: 5′-GAT GCC GAT GAC CTG CAG AAG-3′ (SEQID NO: 282) was hybridized to each of the immobilized templates and theprimer was extended using a commercially available kit (e.g., Sequenaseor Thermosequenase, U.S. Biochemical Corp). The addition of SequenaseDNA polymerase or Thermosequenase DNA polymerase in the presence ofthree deoxyribonucleoside triphosphates (dNTPs; dATP, dGTP, dTTP) anddideoxyribonucleoside cytosine triphosphate (ddCTP) in buffer accordingto the instructions provided by the manufacturer resulted in a singlebase extension of the 21-mer primer bound to the immobilized templateencoding the wildtype apolipoprotein E gene and a three base extensionof the 21-mer primer bound to the immobilized template encoding themutant form of apolipoprotein E gene.

The wafers were analyzed by mass spectrometry as described herein. Thewildtype apolipoprotein E sequence results in a mass spectrum thatdistinguishes the primer with a single base extension (22-mer) with amass to charge ratio of 6771.17 Da (the theoretical mass to charge ratiois 6753.5 Da) from the original 21-mer primer with a mass to chargeration of 6499.64 Da. The mutant apolipoprotein E sequence results in amass spectrum that distinguishes the primer with a three base extension(24-mer) with a mass to charge ratio of 7386.9 (the theoretical masscharge is 7386.9) from the original 21-mer primer with a mass to chargeration of 6499.64 Da.

EXAMPLE 33 Detection of Double-Stranded Nucleic Acid Molecules ViaStrand Displacement and Hybridization to an Immobilized ComplementaryNucleic Acid

This example describes immobilization of a 24-mer primer and thespecific hybridization of one strand of a duplex DNA molecule, therebypermitting amplification of a selected target molecule in solution phaseand permitting detection of the double stranded molecule. This method isuseful for detecting single base changes, and, particularly forscreening genomic libraries of double-stranded fragments.

A 24-mer DNA primer CTGATGCGTC GGATCATCTT TTTT SEQ ID NO: 122,containing a 3′-free thiol group was coupled to a SIAB-derivatizedsilicon wafer as described in Example 29.

An 18-mer synthetic oligonucleotide: 5′-CTGATGCGTCGGATCATC-3′ (SEQ IDNO: 286) was premixed with a 12-mer 5′-GATGATCCGACG-3′ (SEQ ID NO: 285)that has a sequence that is complementary to 12 base portion of the18-mer oligonucleotide. The oligonucleotide mix was heated to 75° C. andcooled slowly to room temperature to facilitate the formation of aduplex molecule:

5′-CTGATGCGTCGGATCATC-3′ (SEQ ID NO: 286) 3′-GCAGCCTAGTAG-5′. (SEQ IDNO: 287)

The specific hybridization of the 12-mer strand of the duplex moleculeto the immobilized 24-mer primer was carried out by mixing 1 μM of theduplex molecule using the hybridization conditions described in Example30.

The wafers were analyzed by mass spectrometry as described above.Specific hybridization was detected in a mass spectrum of the 12-merwith a mass to charge ratio of 3682.78 Da.

EXAMPLE 341-(2-Nitro-5-(3-O-4,4′-dimethoxytritylpropoxy)phenyl)-1-O-((2-cyanoethoxy)-diisopropylaminophosphino)ethaneA. 2-Nitro-5-(3-hydroxypropoxy)benzaldehyde

3-Bromo-1-propanol (3.34 g, 24 mmol) was refluxed in 80 ml of anhydrousacetonitrile with 5-hydroxy-2-nitrobenzaldehyde (3.34 g, 20 mmol), K₂CO₃(3.5 g), and KI (100 mg) overnight (15 h). The reaction mixture wascooled to room temperature and 150 ml of methylene chloride was added.The mixture was filtered and the solid residue was washed with methylenechloride. The combined organic solution was evaporated to dryness andredissolved in 100 ml methylene chloride. The resulted solution waswashed with saturated NaCl solution and dried over sodium sulfate. 4.31g (96%) of desired product was obtained after removal of the solvent invacuo.

R_(f)=0.33 (dichloromethane/methanol, 95/5).

UV (methanol) maximum: 313, 240 (shoulder), 215 nm; minimum: 266 nm.

¹H NMR (DMSO-d₆) δ 10.28 (s, 1H), 8.17 (d, 1H), 7.35 (d, 1H), 7.22 (s,1H), 4.22 (t, 2H), 3.54 (t, 2H), 1.90 (m, 2H).

¹³C NMR (DMSO-d₆) δ189.9, 153.0, 141.6, 134.3, 127.3, 118.4, 114.0,66.2, 56.9, 31.7.

B. 2-Nitro-5-(3-O-t-butyldimethylsilylpropoxy)benzaldehyde

2-Nitro-5-(3-hydroxypropoxy)benzaldehyde (1 g, 4.44 mmol) was dissolvedin 50 ml anhydrous acetonitrile. To this solution, it was added 1 ml oftriethylamine, 200 mg of imidazole, and 0.8 g (5.3 mmol) of tBDMSCl. Themixture was stirred at room temperature for 4 h. Methanol (1 ml) wasadded to stop the reaction. The solvent was removed in vacuo and thesolid residue was redissolved in 100 ml methylene chloride. The resultedsolution was washed with saturated sodium bicarbonate solution and thenwater. The organic phase was dried over sodium sulfate and the solventwas removed in vacuo. The crude mixture was subjected to a quick silicagel column with methylene chloride to yield 1.44 g (96%) of2-nitro-5-(3-O-t-butyldimethylsilylpropoxy)benzaldehyde.

R_(f)=0.67 (hexane/ethyl acetate, 5/1).

UV (methanol), maximum: 317, 243, 215 nm; minimum: 235, 267 nm.

¹H NMR (DMSO-d₆) δ 10.28 (s, 1H), 8.14 (d, 1H), 7.32 (d, 1H), 7.20 (s,1H), 4.20 (t, 2H), 3.75 (t, 2H), 1.90 (m, 2H), 0.85 (s, 9H), 0.02 (s,6H).

¹³C NMR (DMSO-d₆) δ 189.6, 162.7, 141.5, 134.0, 127.1, 118.2, 113.8,65.4, 58.5, 31.2, 25.5, −3.1, −5.7.

C. 1-(2-Nitro-5-(3-O-t-butyldimethylsilylpropoxy)phenyl)ethanol

High vacuum dried2-nitro-5-(3-O-t-butyldimethylsilylpropoxy)benzaldehyde (1.02 g, 3 mmol)was dissolved 50 ml of anhydrous methylene chloride. 2 MTrimethylaluminium in toluene (3 ml) was added dropwise within 10 minand kept the reaction mixture at room temperature. It was stirredfurther for 10 min and the mixture was poured into 10 ml ice cooledwater. The emulsion was separated from water phase and dried over 100 gof sodium sulfate to remove the remaining water. The solvent was removedin vacuo and the mixture was applied to a silica gel column withgradient methanol in methylene chloride. 0.94 g (86%) of desired productwas isolated.

R_(f)=0.375 (hexane/ethyl acetate, 5/1).

UV (methanol), maximum: 306, 233, 206 nm; minimum: 255, 220 nm.

¹H NMR (DMSO-d₆) δ 8.00 (d, 1H), 7.36 (s, 1H), 7.00 (d, 1H), 5.49 (b,OH), 5.31 (q, 1H), 4.19 (m, 2H), 3.77 (t, 2H), 1.95 (m, 2H), 1.37 (d,3H), 0.86 (s, 9H), 0.04 (s, 6H).

¹³C NMR (DMSO-d₆) δ 162.6, 146.2, 139.6, 126.9, 112.9, 112.5, 64.8,63.9, 58.7, 31.5, 25.6, 24.9, −3.4, −5.8.

D. 1-(2-Nitro-5-(3-hydroxypropoxy)phenyl)ethanol

1-(2-Nitro-5-(3-O-t-butyldimethylsilylpropoxy)phenyl)ethanol (0.89 g,2.5 mmol) was dissolved in 30 ml of THF and 0.5 mmol of nBu₄NF was addedunder stirring. The mixture was stirred at room temperature for 5 h andthe solvent was removed in vacuo. The remaining residue was applied to asilica gel column with gradient methanol in methylene chloride.1-(2-Nitro-5-(3-hydroxypropoxy)phenyl)ethanol (0.6 g (99%) was obtained.

R_(f)=0.17 (dichloromethane/methanol, 95/5).

UV (methanol), maximum: 304, 232, 210 nm; minimum: 255, 219 nm.

¹H NMR (DMSO-d₆) δ 8.00 (d, 1H), 7.33 (s, 1H), 7.00 (d, 1H), 5.50 (d,OH), 5.28 (t, OH), 4.59 (t, 1H), 4.17 (t, 2H), 3.57 (m, 2H), 1.89 (m,2H), 1.36 (d, 2H).

¹³C NMR (DMOS-d₆) δ 162.8, 146.3, 139.7, 127.1, 113.1, 112.6, 65.5,64.0, 57.0, 31.8, 25.0.

E. 1-(2-Nitro-5-(3-O-4,4′-dimethoxytritylpropoxy)phenyl)ethanol

1-(2-Nitro-5-(3-hydroxypropoxy)phenyl)ethanol (0.482 g, 2 mmol) wasco-evaporated with anhydrous pyridine twice and dissolved in 20 mlanhydrous pyridine. The solution was cooled in ice-water bath and 750 mg(2.2 mmol) of DMTCl was added. The reaction mixture was stirred at roomtemperature overnight and 0.5 ml methanol was added to stop thereaction. The solvent was removed in vacuo and the residue wasco-evaporated with toluene twice to remove trace of pyridine. The finalresidue was applied to a silica gel column with gradient methanol inmethylene chloride containing drops of triethylamine to yield 0.96 g(89%) of the desired product1-(2-nitro-5-(3-O-4,4′-dimethoxytrityl-propoxy)phenyl)ethanol.

R_(f)=0.50 (dichloromethane/methanol, 99/1).

UV (methanol), maximum: 350 (shoulder), 305, 283, 276 (shoulder), 233,208 nm; minimum: 290, 258, 220 nm.

¹H NMR (DMSO-d₆) δ 8.00 (d, 1H), 6.82-7.42 (ArH), 5.52 (d, OH), 5.32 (m,1H), 4.23 (t, 2H), 3.71 (s, 6H), 3.17 (t, 2H), 2.00 (m, 2H), 1.37 (d,3H).

¹³C NMR (DMOS-d₆) δ 162.5, 157.9, 157.7, 146.1, 144.9, 140.1, 139.7,135.7, 129.5, 128.8, 127.6, 127.5, 127.3, 126.9, 126.4, 113.0, 112.8,112.6, 85.2, 65.3, 63.9, 59.0, 54.8, 28.9, 24.9.

F.1-(2-Nitro-5-(3-O-4,4′-dimethoxytritylpropoxy)phenyl)-1-O-((2-cyanoethoxy)-diisopropylaminophosphino)ethane

1-(2-Nitro-5-(3-O-4,4′-dimethoxytritylpropoxy)phenyl)ethanol (400 mg,0.74 mmol) was dried under high vacuum and was dissolved in 20 ml ofanhydrous methylene chloride. To this solution, it was added 0.5 mlN,N-diisopropylethylamine and 0.3 ml (1.34 mmol) of2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. The reaction mixturewas stirred at room temperature for 30 min and 0.5 ml of methanol wasadded to stop the reaction. The mixture was washed with saturated sodiumbicarbonate solution and was dried over sodium sulfate. The solvent wasremoved in vacuo and a quick silica gel column with 1% methanol inmethylene chloride containing drops of triethylamine yield 510 mg (93%)the desired phosphoramidite.

R_(f)=0.87 (dichloromethane/methanol, 99/1).

EXAMPLE 351-(4-(3-O-4,4′-Dimethoxytritylpropoxy)-3-methoxy-6-nitrophenyl)-1-O-((2-cyanoethoxy)-diisopropylaminophosphino)ethaneA. 4-(3-Hydroxypropoxy)-3-methoxyacetophenone

3-Bromo-1-propanol (53 ml, 33 mmol) was refluxed in 100 ml of anhydrousacetonitrile with 4-hydroxy-3-methoxyacetophenone (5 g, 30 mmol), K₂CO₃(5 g), and KI (300 mg) overnight (15 h). Methylenechloride (150 ml) wasadded to the reaction mixture after cooling to room temperature. Themixture was filtered and the solid residue was washed with methylenechloride. The combined organic solution was evaporated to dryness andredissolved in 100 ml methylene chloride. The resulted solution waswashed with saturated NaCl solution and dried over sodium sulfate. 6.5 g(96.4%) of desired product was obtained after removal of the solvent invacuo.

R_(f)=0.41 (dichloromethane/methanol, 95/5).

UV (methanol), maximum: 304, 273, 227, 210 nm: minimum: 291, 244, 214nm.

¹H NMR (DMSO-d₆) δ 7.64 (d, 1H), 7.46 (s, 1H), 7.04 (d, 1H), 4.58 (b,OH), 4.12 (t, 2H), 3.80 (s, 3H), 3.56 (t, 2H), 2.54 (s, 3H), 1.88 (m,2H).

¹³C NMR (DMSO-d₆) δ 196.3, 152.5, 148.6, 129.7, 123.1, 111.5, 110.3,65.4, 57.2, 55.5, 31.9, 26.3.

B. 4-(3-Acetoxypropoxy)-3-methoxyacetophenone

4-(3-Hydroxypropoxy)-3-methoxyacetophenone (3.5 g, 15.6 mmol) was driedand dissolved in 80 ml anhydrous acetonitrile. This mixture, 6 ml oftriethylamine and 6 ml of acetic anhydride were added. After 4 h, 6 mlmethanol was added and the solvent was removed in vacuo. The residue wasdissolved in 100 ml dichloromethane and the solution was washed withdilute sodium bicarbonate solution, then water. The organic phase wasdried over sodium sulfate and the solvent was removed. The solid residuewas applied to a silica gel column with methylene chloride to yield 4.1g of 4-(3-acetoxypropoxy)-3-methoxyacetophenone (98.6%).

R_(f)=0.22 (dichloromethane/methanol, 99/1).

UV (methanol), maximum: 303, 273, 227, 210 nm; minimum: 290, 243, 214nm.

¹H NMR (DMSO-d₆) δ 7.62 (d, 1H), 7.45 (s, 1H), 7.08 (d, 1H), 4.12 (m,4H, 3.82 (s, 3H), 2.54 (s, 3H), 2.04 (m, 2H), 2.00 (s, 3H).

¹³C NMR (DMSO-d₆) δ 196.3, 170.4, 152.2, 148.6, 130.0, 123.0, 111.8,110.4, 65.2, 60.8, 55.5, 27.9, 26.3, 20.7.

C. 4-(3-Acetoxypropoxy)-3-methoxy-6-nitroacetophenone

4-(3-Acetoxypropoxy)-3-methoxyacetophenone (3.99 g, 15 mmol) was addedportionwise to 15 ml of 70% HNO₃ in water bath and keep the reactiontemperature at the room temperature. The reaction mixture was stirred atroom temperature for 30 min and 30 g of crushed ice was added. Thismixture was extracted with 100 ml of dichloromethane and the organicphase was washed with saturated sodium bicarbonate solution. Thesolution was dried over sodium sulfate and the solvent was removed invacuo. The crude mixture was applied to a silica gel column withgradient methanol in methylene chloride to yield 3.8 g (81.5%) ofdesired product 4-(3-acetoxypropoxy)-3-methoxy-6-nitroacetophenone and0.38 g (8%) of ipso-substituted product5-(3-acetoxypropoxy)-4-methoxy-1,2-dinitrobenzene.

Side ipso-substituted product5-(3-acetoxypropoxy)-4-methoxy-1,2-dinitrobenzene:

R_(f)=0.47 (dichloromethane/methanol, 99/1).

UV (methanol), maximum: 334, 330, 270, 240, 212 nm; minimum: 310, 282,263, 223 nm.

¹H NMR (CDCl₃) δ 7.36 (s, 1H), 7.34 (s, 1H), 4.28 (t, 2H), 4.18 (t, 2H),4.02 (s, 3H), 2.20 (m, 2H), 2.08 (s, 3H).

¹³C NMR (CDCl³) δ 170.9, 152.2, 151.1, 117.6, 111.2, 107.9, 107.1, 66.7,60.6, 56.9, 28.2, 20.9.

Desired product 4-(3-acetoxypropoxy)-3-methoxy-6-nitroacetophenone:

R_(f)=0.29 (dichloromethane/methanol, 99/1).

UV (methanol), maximum: 344, 300, 246, 213 nm; minimum: 320, 270, 227nm.

¹H NMR (CDCl₃) δ 7.62 (s, 1H), 6.74 (s, 1H), 4.28 (t, 2H), 4.20 (t, 2H),3.96 (s, 3H), 2.48 (s, 3H), 2.20 (m, 2H), 2.08 (s, 3H).

¹³C NMR (CDCl₃) δ 200.0, 171.0, 154.3, 148.8, 138.3, 133.0, 108.8,108.0, 66.1, 60.8, 56.6, 30.4, 28.2, 20.9.

D. 1-(4-(3-Hydroxypropoxy)-3-methoxy-6-nitrophenyl)ethanol

4-(3-Acetoxypropoxy)-3-methoxy-6-nitroacetophenone (3.73 g, 12 mmol) wasadded 150 ml ethanol and 6.5 g of K₂CO₃. The mixture was stirred at roomtemperature for 4 h and TLC with 5% methanol in dichloromethaneindicated the completion of the reaction. To this same reaction mixture,it was added 3.5 g of NaBH₄ and the mixture was stirred at roomtemperature for 2 h. Acetone (10 ml) was added to react with theremaining NaBH₄. The solvent was removed in vacuo and the residue wasuptaken into 50 g of silica gel. The silica gel mixture was applied onthe top of a silica gel column with 5% methanol in methylene chloride toyield 3.15 g (97%) of desired product1-(4-(3-hydroxypropoxy)-3-methoxy-6-nitrophenyl)ethanol.

Intermediate product 4-(3-hydroxypropoxy)-3-methoxy-6-nitroacetophenoneafter deprotection:

R_(f)=0.60 (dichloromethane/methanol, 95/5).

Final product 1-(4-(3-hydroxypropoxy)-3-methoxy-6-nitrophenyl)ethanol:

R_(f)=0.50 (dichloromethane/methanol, 95/5).

UV (methanol), maximum: 344, 300, 243, 219 nm: minimum: 317, 264, 233nm.

¹H NMR (DMSO-d₆) δ 7.54 (s, 1H), 7.36 (s, 1H), 5.47 (d, OH), 5.27 (m,1H), 4.55 (t, OH), 4.05 (t, 2H), 3.90 (s, 3H), 3.55 (q, 2H), 1.88 (m,2H), 1.37 (d, 3H).

¹³C NMR (DMSO-d₆) δ 153.4, 146.4, 138.8, 137.9, 109.0, 108.1, 68.5,65.9, 57.2, 56.0, 31.9, 29.6.

E.1-(4-(3-O-4,4′-Dimethoxytritylpropoxy)-3-methoxy-6-nitrophenyl)ethanol

1-(4-(3-Hydroxypropoxy)-3-methoxy-6-nitrophenyl)ethanol (0.325 g, 1.2mmol) was co-evaporated with anhydrous pyridine twice and dissolved in15 ml anhydrous pyridine. The solution was cooled in ice-water bath and450 mg (1.33 mmol) of DMTCl was added. The reaction mixture was stirredat room temperature overnight and 0.5 ml methanol was added to stop thereaction. The solvent was removed in vacuo and the residue wasco-evaporated with toluene twice to remove trace of pyridine. The finalresidue was applied to a silica gel column with gradient methanol inmethylene chloride containing drops of triethylamine to yield 605 mg(88%) of desired product1-(4-(3-O-4,4′-dimethoxytritylpropoxy)-3-methoxy-6-nitrophenyl)ethanol.

R_(f)=0.50 (dichloromethane/methanol, 95/5).

UV (methanol), maximum: 354, 302, 282, 274, 233, 209 nm; minimum: 322,292, 263, 222 nm.

¹H NMR (DMSO-d₆) δ 7.54 (s, 1H), 6.8-7.4 (ArH), 5.48 (d, OH), 5.27 (m,1H), 4.16 (t, 2H), 3.85 (s, 3H), 3.72 (s, 6H), 3.15 (t, 2H), 1.98 (t,2H), 1.37 (d, 3H).

¹³C NMR (DMSO-d₆) δ 157.8, 153.3, 146.1, 144.9, 138.7, 137.8, 135.7,129.4, 128.7, 127.5, 127.4, 126.3, 112.9, 112.6, 108.9, 108.2, 85.1,65.7, 63.7, 59.2, 55.8, 54.8, 29.0, 25.0.

F.1-(4-(3-O-4,4′-Dimethoxytritylpropoxy)-3-methoxy-6-nitrophenyl)-1-O-((2-cyanoethoxy)-diisopropylaminophosphino)ethane

1-(4-(3-O-4,4′-Dimethoxytritylpropoxy)-3-methoxy-6-nitrophenyl)ethanol(200 mg, 3.5 mmol) was dried under high vacuum and was dissolved in 15ml of anhydrous methylene chloride. To this solution, it was added 0.5ml N,N-diisopropylethylamine and 0.2 ml (0.89 mmol) of2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. The reaction mixturewas stirred at room temperature for 30 min and 0.5 ml of methanol wasadded to stop the reaction. The mixture was washed with saturated sodiumbicarbonate solution and was dried over sodium sulfate. The solvent wasremoved in vacuo and a quick silica gel column with 1% methanol inmethylene chloride containing drops of triethylamine yield 247 mg(91.3%) the desired phosphoramidite1-(4-(3-0-4,4′-dimethoxytritylpropoxy)-3-methoxy-6-nitrophenyl)-1-O-((2-cyanoethoxy)-diisopropylaminophosphino)ethane.

R_(f)=0.87 (dichloromethane/methanol, 99/1).

EXAMPLE 36 Oligonucleotide Synthesis

The oligonucleotide conjugates containing photocleavable linker wereprepared by solid phase nucleic acid synthesis (see: Sinha et al.Tetrahedron Lett. 1983, 24: 5843-5846; Sinha et al. Nucleic Acids Res.1984, 12: 4539-4557; Beaucage et al. Tetrahedron 1993, 49: 6123-6194;and Matteucci et al. J. Am. Chem. Soc. 1981, 103: 3185-3191) understandard conditions. In addition a longer coupling time period wasemployed for the incorporation of photocleavable unit and the 5′terminal amino group. The coupling efficiency was detected by measuringthe absorbance of released DMT cation and the results indicated acomparable coupling efficiency of phosphoramidite1-(2-nitro-5-(3-O-4,4′-dimethoxytritylpropoxy)phenyl)-1-O-((2-cyanoethoxy)-diisopropylaminophosphino)ethaneor1-(4-(3-O-4,4′-dimethoxytritylpropoxy)-3-methoxy-6-nitrophenyl)-1-O-((2-cyanoethoxy)-diisopropylaminophosphino)ethanewith those of common nucleoside phosphoramodites. Deprotection of thebase protection and release of the conjugates from the solid support wascarried out with concentrated ammonium at 55° C. overnight. Deprotectionof the base protection of other conjugates was done by fast deprotectionwith AMA reagents. Purification of the MMT-on conjugates was done byHPLC (trityl-on) using 0.1 M triethylammonium acetate, pH 7.0 and agradient of acetonitrile (5% to 25% in 20 minutes). The collected MMT orDMT protected conjugate was reduced in volume, detritylated with 80%aqueous acetic acid (40 min, 0° C.), desalted, stored at −20° C.

EXAMPLE 37 Photolysis Study

In a typical case, 2 nmol of oligonucleotide conjugate containingphotocleavable linker in 200 μl distilled water was irradiated with along wavelength UV lamp (Blak Ray XX-15 UV lamp, Ultraviolet products,San Gabriel, Calif.) at a distance of 10 cm (emission peak 365 nm, lampintensity=1.1 mW/cm² at a distance of 31 cm). The resulting mixture wasanalyzed by HPLC (trityl-off) using 0.1 M triethylammonium acetate, pH7.0 and a gradient of acetonitrile. Analysis showed that the conjugatewas cleaved from the linker within minutes upon UV irradiation.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims.

1-103. (canceled)
 104. A process for characterizing the structure of one or more target nucleic acids from a biological sample, comprising: a) determining the base composition of at least one nucleic acid molecule having a known sequence; b) amplifying one or more target nucleic acid molecules from a biological sample; c) ionizing and volatilizing the product of step b); d) analyzing a product of step c) by mass spectrometry to determine an observed molecular mass of said product of step c); e) obtaining a number of possible base compositions of said product of step c) from the observed molecular mass determined in step d); f) constraining the number of possible base compositions obtained in step e); and g) comparing at least one base composition obtained in step e) or at least one base composition constrained in step f) with the base composition determined in step a); whereby the structure of the one or more target nucleic acids is characterized based on the comparison of (g).
 105. The process of claim 104 wherein step d) further comprises analyzing a second product of step c) by mass spectrometry to determine an observed molecular mass of the second product of step c), wherein said second product of step c) is the amplification product complementary to the product of step c), and wherein step e) further comprises obtaining a number of possible base compositions of the second product of step c) from the observed molecular mass of the second product of step c), and wherein step f) further comprises constraining the number of possible base compositions of the second product of step c).
 106. The process of claim 105 wherein step f) comprises constraining the number of possible base compositions of said product of step c) using a composition constraint, said composition constraint comprising the base composition obtained in step e) for the second product of step c), or the base composition constrained in step f) for the second product of step c).
 107. The process of claim 104 wherein step f) comprises making a mass measurement with an accuracy allowing detection of a mass shift of 0.03% to 0.005%.
 108. The process of claim 104 wherein electrospray mass spectrometry is used in step d).
 109. The process of claim 104 wherein step f) comprises the use of a composition constraint.
 110. The process of claim 109 wherein said composition constraint comprises the length of the product of step c).
 111. The process of claim 109 wherein said composition constraint comprises at least one known nucleotide in the sequence of the product of step c).
 112. The process of claim 111 wherein said known nucleotide is a nucleotide in the sequence of an amplification primer used to generate the product of step c).
 113. The process of claim 109 wherein said composition constraint comprises sequence information of a nucleic acid complementary to the product of step c).
 114. The process of claim 113 wherein said sequence information comprises at least one known nucleotide in the sequence of the nucleic acid complementary to the product of step c).
 115. The process of claim 113 wherein said composition constraint comprises the base composition of a second product of step c), wherein said second product of step c) is the amplification product complementary to the product of step c).
 116. The process of claim 104, wherein amplification step b) is performed by PCR using primers complementary to sequences of the target nucleic acid molecule.
 117. The process of claim 116, wherein step f) comprises the use of a composition constraint, said composition constraint comprising sequence information from a nucleic acid molecule known to be amplifiable using the primers complementary to sequences of the target nucleic acid molecule.
 118. The process of claim 104, wherein said determining in step a) comprises calculating the base composition of the at least one nucleic acid molecule having a known sequence.
 119. The process of claim 104, wherein said characterizing the structure of one or more target nucleic acids in a biological sample comprises determining the molecular mass of one or more target nucleic acids in a biological sample.
 120. The process of claim 104, wherein said characterizing the structure of one or more target nucleic acids in a biological sample comprises determining the base composition of one or more target nucleic acids in a biological sample.
 121. The process of claim 104, wherein said characterizing the structure of one or more target nucleic acids in a biological sample comprises determining the length of one or more target nucleic acids in a biological sample.
 122. A process for characterizing the structure of one or more target nucleic acids from a biological sample, comprising: a) determining the base composition and molecular mass of at least one nucleic acid molecule having a known sequence; b) amplifying one or more target nucleic acid molecules from a biological sample; c) ionizing and volatilizing the product of step b); d) analyzing a product of step c) by mass spectrometry to determine an observed molecular mass for said product of step c); e) obtaining a number of possible base compositions of said product of step c) from the observed molecular mass determined in step d); f) constraining the number of possible base compositions obtained in step e); and g) comparing at least one base composition obtained in step e) with the base composition determined in step a), or comparing at least one base composition obtained in step f) with the base composition determined in step a), or comparing the observed molecular mass determined in step d) with the molecular mass determined in step a); whereby the structure of the one or more target nucleic acids is characterized based on the comparison of (g).
 123. The process of claim 122 wherein step d) further comprises analyzing a second product of step c) by mass spectrometry to determine an observed molecular mass of the second product of step c), wherein said second product of step c) is the amplification product complementary to the product of step c), and wherein step e) further comprises obtaining a number of possible base compositions of the second product of step c) from the observed molecular mass of the second product of step c), and wherein step f) further comprises constraining the number of possible base compositions of the second product of step c).
 124. The process of claim 123 wherein step f) comprises constraining the number of possible base compositions of said product of step c) using a composition constraint, said composition constraint comprising the base composition obtained in step e) for the second product of step c), or the base composition constrained in step f) for the second product of step c).
 125. The process of claim 122 wherein step f) comprises making a mass measurement with an accuracy allowing detection of a mass shift of 0.03% to 0.005%.
 126. The process of claim 122 wherein electrospray mass spectrometry is used in step d).
 127. The process of claim 122 wherein step f) comprises the use of a composition constraint.
 128. The process of claim 127 wherein said composition constraint comprises the length of the product of step c).
 129. The process of claim 127 wherein said composition constraint comprises at least one known nucleotide in the sequence of the product of step c).
 130. The process of claim 129 wherein said known nucleotide is a nucleotide in the sequence of an amplification primer used to generate the product of step c).
 131. The process of claim 127 wherein said composition constraint comprises sequence information of a nucleic acid complementary to the product of step c).
 132. The process of claim 131 wherein said sequence information comprises at least one known nucleotide in the sequence of the nucleic acid complementary to the product of step c).
 133. The process of claim 131 wherein said composition constraint comprises the base composition of a second product of step c), wherein said second product of step c) is the amplification product complementary to the product of step c).
 134. The process of claim 122, wherein said amplification step b) is performed using PCR with primers complementary to sequences of the target nucleic acid molecule.
 135. The process of claim 134, wherein step f) comprises the use of a composition constraint, said composition constraint comprising sequence information from a nucleic acid molecule known to be amplifiable using the primers complementary to sequences of the target nucleic acid molecule.
 136. The process of claim 122, wherein said determining in step a) comprises calculating the base composition and molecular mass of the at least one nucleic acid molecule having a known sequence.
 137. The process of claim 122, wherein said characterizing the structure of one or more target nucleic acids in a biological sample comprises determining the molecular mass of one or more target nucleic acids in a biological sample.
 138. The process of claim 122, wherein said characterizing the structure of one or more target nucleic acids in a biological sample comprises determining the base composition of one or more target nucleic acids in a biological sample.
 139. The process of claim 122, wherein said characterizing the structure of one or more target nucleic acids in a biological sample comprises determining the length of one or more target nucleic acids in a biological sample. 